Nucleic acid compositions for stimulating immune responses

ABSTRACT

The invention provides an immunostimulatory nucleic acid comprising CpG motifs, and methods of use thereof in stimulating immunity.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 60/394,091, entitled “NUCLEIC ACIDCOMPOSITIONS FOR STIMULATING IMMUNE RESPONSES”, filed on Jul. 3, 2002,which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to immunostimulatory nucleicacids, compositions thereof and methods of using the immunostimulatorynucleic acids.

BACKGROUND OF THE INVENTION

Bacterial DNA has immune stimulatory effects to activate B cells andnatural killer cells, but vertebrate DNA does not (Tokunaga, T., et al.,1988. Jpn. J. Cancer Res. 79:682-686; Tokunaga, T., et al., 1984, JNCI72:955-962; Messina, J. P., et al., 1991, J. Immunol. 147:1759-1764; andreviewed in Krieg, 1998, In: Applied Oligonucleotide Technology, C. A.Stein and A. M. Krieg, (Eds.), John Wiley and Sons, Inc., New York,N.Y., pp. 431-448). It is now understood that these immune stimulatoryeffects of bacterial DNA are a result of the presence of unmethylatedCpG dinucleotides in particular base contexts (CpG motifs), which arecommon in bacterial DNA, but methylated and underrepresented invertebrate DNA (Krieg et al, 1995 Nature 374:546-549; Krieg, 1999Biochim. Biophys. Acta 93321:1-10).

The immune stimulatory effects of bacterial DNA can be mimicked withsynthetic oligodeoxynucleotides (ODN) containing these CpG motifs. SuchCpG ODN have highly stimulatory effects on human and murine leukocytes,inducing B cell proliferation; cytokine and immunoglobulin secretion;natural killer (NK) cell lytic activity and IFN-γ secretion; andactivation of dendritic cells (DCs) and other antigen presenting cellsto express costimulatory molecules and secrete cytokines, especially theTh1-like cytokines that are important in promoting the development ofTh1-like T cell responses. These immune stimulatory effects of nativephosphodiester backbone CpG ODN are highly CpG specific in that theeffects are essentially abolished if the CpG motif is methylated,changed to a GpC, or otherwise eliminated or altered (Krieg et al, 1995Nature 374:546-549; Hartmann et al, 1999 Proc. Natl. Acad. Sci USA96:9305-10). Phosphodiester CpG ODN can be formulated in lipids, alum,or other types of vehicles with depot properties or improved cell uptakein order to enhance the immune stimulatory effects (Yamamoto et al, 1994Microbiol. Immunol. 38:831-836; Gramzinski et al, 1998 Mol. Med.4:109-118).

In early studies, it was thought that the immune stimulatory CpG motiffollowed the formula purine-purine-CpG-pyrimidine-pyrimidine (Krieg etal, 1995 Nature 374:546-549; Pisetsky, 1996 J. Immunol. 156:421-423;Hacker et al., 1998 EMBO J. 17:6230-6240; Lipford et al., 1998 Trends inMicrobiol. 6:496-500). However, it is now clear that mouse lymphocytesrespond quite well to phosphodiester CpG motifs that do not follow this“formula” (Yi et al., 1998 J. Immunol. 160:5898-5906) and the same istrue of human B cells and dendritic cells (Hartmann et al, 1999 Proc.Natl. Acad. Sci USA 96:9305-10; Liang, 1996 J. Clin. Invest.98:1119-1129).

Several past investigators have looked at whether the nucleotide contentof ODN may have effects independently of the sequence of the ODN.Interestingly, antisense ODN have been found to be generally enriched inthe content of GG, CCC, CC, CAC, and CG sequences, while having reducedfrequency of TT or TCC nucleotide sequences compared to what would beexpected if base usage were random (Smetsers et al., 1996 AntisenseNucleic Acid Drug Develop. 6:63-67). This raised the possibility thatthe over-represented sequences may comprise preferred targeting elementsfor antisense oligonucleotides or visa versa. One reason to avoid theuse of thymidine-rich ODN for antisense experiments is that degradationof the ODN by nucleases present in cells releases free thymidine whichcompetes with ³H-thymidine which is frequently used in experiments toassess cell proliferation (Matson et al., 1992 Antisense Research andDevelopment 2:325-330).

SUMMARY OF THE INVENTION

The invention is based in part on the surprising discovery that a newfamily of nucleic acids that induce higher levels of immune stimulationthan previously known nucleic acids. This finding was surprising in partbecause more than 100 nucleic acid sequences were screened prior todiscovering those disclosed herein.

The invention provides in one aspect, a composition comprising animmunostimulatory nucleic acid molecule comprising the nucleotidesequence of SEQ ID NO:1.

The invention further provides in another aspect, a method forstimulating an immune response in a subject in need thereof comprisingadministering to a subject an immunostimulatory nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, in an amounteffective to stimulate an immune response.

Various embodiments of the invention apply equally to the aspectsprovided herein and some of these are recited below.

In one embodiment, the immunostimulatory nucleic acid molecule consistsof the nucleotide sequence of SEQ ID NO:1.

In another embodiment, the composition further comprises an antigen.Alternatively, the subject to be treated is further administered anantigen. The antigen may be selected from the group consisting of amicrobial antigen, a self antigen, a cancer antigen, and an allergen,but it is not so limited. In one embodiment, the microbial antigen isselected from the group consisting of a bacterial antigen, a viralantigen, a fungal antigen and a parasitic antigen. In anotherembodiment, the antigen is encoded by a nucleic acid vector. In arelated embodiment, the nucleic acid vector is separate from theimmunostimulatory nucleic acid. The antigen may be a peptide antigen.

In another embodiment, the composition further comprises an adjuvant, orthe subject is further administered an adjuvant. The adjuvant may be amucosal adjuvant, but it is not so limited.

In another embodiment, the composition further comprises a cytokine, orthe subject is further administered a cytokine.

In still another embodiment, the composition further comprises atherapeutic agent selected from the group consisting of ananti-microbial agent, an anti-cancer agent, and an allergy/asthmamedicament, or the subject is further administered a therapeutic agentselected from the same group. In a related embodiment, theanti-microbial agent is selected from the group consisting of ananti-bacterial agent, an anti-viral agent, an anti-fungal agent, and ananti-parasite agent. In another related embodiment, the anti-canceragent is selected from the group consisting of a chemotherapeutic agent,a cancer vaccine, and an immunotherapeutic agent. In still anotherrelated embodiment, the allergy/asthma medicament is selected from thegroup consisting of PDE-4 inhibitor, bronchodilator/beta-2 agonist, K+channel opener, VLA-4 antagonist, neurokin antagonist, TXA2 synthesisinhibitor, xanthanine, arachidonic acid antagonist, 5 lipoxygenaseinhibitor, thromboxin A2 receptor antagonist, thromboxane A2 antagonist,inhibitor of 5-lipox activation protein, and protease inhibitor.

The immunostimulatory nucleic acid may in some embodiments have anucleotide backbone which includes at least one backbone modification.In one embodiment, the backbone modification is a phosphorothioatemodification. In another embodiment, the nucleotide backbone ischimeric. In one embodiment, the nucleotide backbone is entirelymodified.

In one embodiment, the composition further comprises a pharmaceuticallyacceptable carrier.

In one embodiment, the immunostimulatory nucleic acid is free ofmethylated CpG dinucleotides. In another embodiment, theimmunostimulatory nucleic acid includes at least four CpG motifs. In yetanother embodiment, the immunostimulatory nucleic acid is T-rich. In arelated embodiment, the immunostimulatory nucleic acid includes a poly-Tsequence. In another embodiment, the immunostimulatory nucleic acidincludes a poly-G sequence.

In certain embodiments, the immunostimulatory nucleic acid is formulatedin a variety of ways. In one embodiment, the immunostimulatory nucleicacid is formulated for oral administration. The immunostimulatorynucleic acid may also be formulated as a nutritional supplement. In arelated embodiment, the nutritional supplement is formulated as acapsule, a pill, or a sublingual tablet. In another embodiment, theimmunostimulatory nucleic acid is formulated for local administration.The immunostimulatory nucleic acid may also be formulated for parenteraladministration or it may be formulated in a sustained release device.The sustained release device may be a microparticle but it is not solimited. In another embodiment, the immunostimulatory nucleic acid isformulated for delivery to a mucosal surface. The mucosal surface may beselected from the group consisting of an oral, nasal, rectal, vaginal,and ocular surface, but is not so limited.

In one embodiment, the immunostimulatory nucleic acid stimulates amucosal immune response. In another embodiment, the immunostimulatorynucleic acid stimulates a systemic immune response. In importantembodiments, the immunostimulatory nucleic acid stimulates both amucosal and systemic immune response. The immune response is anantigen-specific immune response, in some embodiments. In relatedembodiments, the immunostimulatory nucleic acid is provided in an amounteffective to stimulate a mucosal immune response. In other embodiments,the immunostimulatory nucleic acid is provided in an amount effective tostimulate a systemic immune response. In still other embodiments, theimmunostimulatory nucleic acid is provided in an amount effective tostimulate an innate immune response.

In various embodiments, the immunostimulatory nucleic acid is intendedfor treatment or prevention of a variety of diseases. Thus, in oneembodiment, the immunostimulatory nucleic acid is provided in an amounteffective to treat or prevent an infectious disease. In anotherembodiment, the immunostimulatory nucleic acid is provided in an amounteffective to treat or prevent an allergy. In still another embodiment,the immunostimulatory nucleic acid is provided in an amount effective totreat or prevent asthma. In yet a further embodiment, theimmunostimulatory nucleic acid is provided in an amount effective totreat or prevent a cancer.

In a related embodiment, the infectious disease is a herpes simplexvirus infection. In another embodiment, the immunostimulatory nucleicacid is intended for administration to a subject that has or is at riskof developing an infection. The infection may be selected from the groupconsisting of a bacterial infection, a viral infection, a fungalinfection, and a parasite infection. In one embodiment, the viralinfection is selected from the group consisting of Humanimmunodeficiency viruses (HIV-1 and HIV-2), Human T lymphotrophic virustype I (HTLV-I), Human T lymphotrophic virus type II (HTLV-II), Herpessimplex virus type I (HSV-1), Herpes simplex virus type 2 (HSV-2), Humanpapilloma virus (multiple types), Hepatitis A virus, Hepatitis B virus,Hepatitis C and D viruses, Epstein-Barr virus (EBV), Cytomegalovirus andMolluscum contagiosum virus. In an important embodiment, the viralinfection is a herpes simplex virus infection.

In other embodiments, the infection is an infection with a microbialspecies selected from the group consisting of herpesviridae,retroviridae, orthomyroviridae, toxoplasma, haemophilus, campylobacter,clostridium, E. coli, and staphylococcus. In related embodiments, theantigen to be administered to the subject or to be included in thecomposition is from one of the foregoing species.

In other embodiments, the immunostimulatory nucleic acid is intendedfrom administration to a subject that has or is at risk of developingallergy, or a subject that has or is at risk of developing asthma, or asubject that has or is at risk of developing a cancer.

In embodiments relating to the treatment of a subject, the method mayfurther comprise isolating an immune cell from the subject, contactingthe immune cell with an effective amount to activate the immune cell ofthe immunostimulatory nucleic acid and re-administering the activatedimmune cell to the subject. In one embodiment, the immune cell is aleukocyte. In another embodiment, the immune cell is a dendritic cell.In another embodiment, the method further comprises contacting theimmune cell with an antigen.

In important embodiments, the subject is a human. In other embodiments,the subject is selected from the group consisting of a dog, cat, horse,cow, pig, sheep, goat, chicken, monkey and fish.

Accordingly, the methods provided herein can be used on a subject thathas or is at risk of developing an infectious disease and therefore themethod is a method for treating or preventing the infectious disease.The methods can also be used on a subject that has or is at risk ofdeveloping asthma and the method is a method of treating or preventingasthma in the subject. The method can also be used on a subject that hasor is at risk of developing allergy and the method is a method oftreating or preventing allergy. And it can further be used on a subjectthat has or is at risk of developing a cancer and the method is a methodof treating or preventing the cancer. In one embodiment, the cancer isselected from the group consisting of biliary tract cancer; bone cancer;brain and CNS cancer; breast cancer; cervical cancer; choriocarcinoma;colon cancer; connective tissue cancer; endometrial cancer; esophagealcancer; eye cancer; gastric cancer; Hodgkin's lymphoma; intraepithelialneoplasms; larynx cancer; lymphomas; liver cancer; lung cancer (e.g.small cell and non-small cell); melanoma; neuroblastomas; oral cavitycancer; ovarian cancer; pancreas cancer; prostate cancer; rectal cancer;sarcomas; skin cancer; testicular cancer; thyroid cancer; and renalcancer.

In yet another embodiment of the therapeutic or prophylactic methodsprovided herein, the method may further comprise administering anantibody specific for a cell surface antigen, and wherein the immuneresponse results in antigen dependent cellular cytotoxicity (ADCC).

The invention provides in another aspect, a method for preventingdisease in a subject, comprising administering to the subject animmunostimulatory nucleic acid on a regular basis to prevent disease inthe subject, wherein the immunostimulatory nucleic acid has a nucleotidesequence comprising SEQ ID NO:1.

In yet another aspect, the invention provides a method for inducing aninnate immune response, comprising administering to the subject animmunostimulatory nucleic acid in an amount effective for activating aninnate immune response, wherein the immunostimulatory nucleic acid has anucleotide sequence comprising SEQ ID NO:1.

In still another aspect, the invention provides a method for identifyingan immunostimulatory nucleic acid comprising measuring a control levelof activation of an immune cell population contacted with animmunostimulatory nucleic acid comprising a nucleotide sequence of SEQID NO:1, measuring a test level of activation of an immune cellpopulation contacted with a test nucleic acid, and comparing the controllevel of activation to the test level of activation, wherein a testlevel that is equal to or above the control level is indicative of animmunostimulatory nucleic acid.

These and other aspects and embodiments of the invention will bedescribed in greater detail herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: TLR9 engagement by ODNs 7909 and 10103. A TLR9-expressing cellline was incubated with the indicated concentrations of ODNs asdescribed in Materials and Methods. Shown is the mean stimulation indexabove media control for 4 independent experiments. IL-1 was used as apositive control for the reporter gene.

FIG. 2: B cells up regulate the activation marker CD86 upon incubationof PBMC with CpG ODNs. Human PBMC were incubated with ODNs 7909 and10103 as well as a control ODN at the indicated concentrations for 48 h.Shown is the mean percentage of CD86 expressing CD19-positive B cells(measured by flow cytometry) of three different donors.

FIG. 3: Proliferation of B cells induced by CpG ODNs 7909 and 10103.PBMC pre-incubated with the dye CFSE were cultured for 5 days without orwith the indicated ODN concentrations. Cells were harvested and thedecrease of the CFSE stain on proliferating CD19-positive B cells wasmeasured by flow cytometry (see also Materials and Methods).

FIG. 4: IFN-α secretion induced by ODNs 7909 and 10103. Human PBMC ofsix different donors were incubated with the indicated concentrations ofODNs for 48 h. The supernatant was harvested and IFN-α was measured byELISA (see Materials and Methods). Shown are the amounts of IFN-αobtained for the six different donors at each concentration.

FIG. 5: IP-10 secretion induced by ODNs 7909 and 10103. Human PBMC ofthree different donors were incubated with the indicated concentrationsof ODNs for 48 h. The supernatant was harvested and IP-10 was measuredby ELISA (see Materials and Methods). Shown are the mean amounts ofIP-10 obtained for the three different donors at each concentration.

FIG. 6: showed the secretion of IL-10 upon incubation with differentconcentrations of 7909, 10103 and control ODN. Shown are the means fromthree different donors obtained upon incubation for 48 h as indicated.

FIG. 7: TNF-α secretion: PBMC of three different blood donors wereincubated with the indicated concentrations of ODNs 7909, 10103 or acontrol for 48 h. Supernatants were harvested and TNF-α was measured byELISA. Shown are the mean amounts for three donors.

FIG. 8: Naïve BALB/c mouse splenocytes (5×10⁶/ml or 2.5×10⁶/ml) wereincubated with media (negative control) or different amounts of CpG ODN7909 (white bars), 10103 (black bars). Cells were pulsed with³H-thymidine (20 μCi/ml) at 96 hr post incubation for 16 hours,harvested and measured for radioactivity. Each bar represents thestimulation index (counts/min (CPM) of cells incubated/CPM of cellsincubated with media).

FIG. 9: Naïve BALB/c mouse splenocytes (5×10⁶/ml) were incubated withmedia (negative control) or different amounts of CpG ODN 7909, 10103 orcontrol ODN 2137. Supernatants were harvested at 6 hr (for TNF-α, panelD), 24 hr (IL-12, panel B) or 48 hr (for IL-6, panel C, and IL-10, panelA).

FIG. 10: Naïve BALB/c mouse splenocytes (30×10⁶/ml) were incubated withmedia (negative control) or different amounts of CpG ODN 7909 and 10103.NK activity was measured by using ⁵¹Cr release assay.

FIG. 11: Adult (6-8 wk) BALB/c mice were immunized with 1 μg of HBsAgalone or in combination with CpG ODN (10 μg) 10103, 7909 or control ODN(10 μg) 2137. Animals were bled at 4 weeks post immunization and plasmawas assayed for total IgG levels against HBsAg (Anti-HBs). Each barrepresents the geometric mean (±SEM) of the ELISA end point dilutiontiter for the entire group (n=5). Titers were defined as the highestdilution resulting in an absorbance value two times that of non-immuneplasma with a cut-off value of 0.05.

FIG. 12: Adult BALB/c mice (6-8 wks old) were immunized with 1 mg ofHBsAg alone or in combination with 10 mg CpG ODN 7909, 10103 or 10 μgcontrol ODN 2137. Animals were bled at 4 weeks post immunization andplasma was assayed for IgG1 and IgG2a levels against HBsAg (Anti-HBs).Each bar represents the geometric mean (±SEM) of the ELISA end pointdilution titer for the entire group (n=5). Titers were defined as thehighest dilution resulting in an absorbance value two times that ofnon-immune plasma with a cut-off value of 0.05.

FIG. 13: Adult (6-8 wk) BALB/c mice were immunized with 1 mg of HBsAg incombination with either CpG ODN (10 μg) 7909 or 10103. At 4 weeks postimmunization, spleens were removed and splenocytes were used formeasuring CTL activity by ⁵¹Cr release assay. CTL activity is indicatedas mean % specific lysis (±SEM) for the group of animals (n=5) atdifferent effector:target ratios.

DETAILED DESCRIPTION OF THE INVENTION

It was known in the prior art that CpG containing nucleic acidsstimulate the immune system, and that can thereby be used to treatcancer, infectious diseases, allergy, asthma and other disorders, and tohelp protect against opportunistic infections following cancerchemotherapies. The strong yet balanced, cellular and humoral immuneresponses that result from CpG stimulation reflect the body's ownnatural defense system against invading pathogens and cancerous cells.CpG sequences, while relatively rare in human DNA, are commonly found inthe DNA of infectious organisms such as bacteria. The human immunesystem has apparently evolved to recognize CpG sequences as an earlywarning sign of infection, and to initiate an immediate and powerfulimmune response against invading pathogens without causing adversereactions frequently seen with other immune stimulatory agents. Thus,CpG containing nucleic acids, relying on this innate immune defensemechanism, can utilize a unique and natural pathway for immune therapy.

The effects of CpG nucleic acids on immune modulation were discovered bythe inventor of the instant patent application and have been describedextensively in co-pending patent applications, such as U.S. patentapplication Ser. No. 08/386,063 filed on Feb. 7, 1995 (and related PCTUS95/01570); Ser. No. 08/738,652 filed on Oct. 30, 1996; Ser. No.08/960,774 filed on Oct. 30, 1997 (and related PCT/US97/19791, WO98/18810); Ser. No. 09/191,170 filed on Nov. 13, 1998; Ser. No.09/030,701 filed on Feb. 25, 1998 (and related PCT/US98/03678; Ser. No.09/082,649 filed on May 20, 1998 (and related PCT/US98/10408); Ser. No.09/325,193 filed on Jun. 3, 1999 (and related PCT/US98/04703); Ser. No.09/286,098 filed on Apr. 2, 1999 (and related PCT/US99/07335); Ser. No.09/306,281 filed on May 6, 1999 (and related PCT/US99/09863). The entirecontents of each of these patents and patent applications is herebyincorporated by reference.

The invention is based, in part, on the unexpected discovery of a familyof nucleic acids that is as immunostimulatory as previously reported CpGnucleic acids. This family of nucleic acids comprises the nucleotidesequence having the formula of5′ X₁X₂X₃ X₄X₅X₆ X₇X₈X₉ X₁₀X₁₁X₁₂ GGT CGT TTT 3′ (SEQ ID NO:3)wherein X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₉, X₁₀, X₁₁, and X₁₂ areindependently selected residues that may be selected from the group ofnucleotides consisting of adenosine, guanosine, thymidine, and cytosine.In some embodiments, there may be no flanking residues. Such a nucleicacid would comprise a nucleotide sequence of 5′ GGT CGT TTT 3′ (SEQ IDNO:4).

In other embodiments, the nucleic acid may lack X₁; X₁ and X₂; X₁, X₂and X₃; X₁, X₂, X₃ and X₄; or X₁, X₂, X₃, X₄ and X₅, X₁ through X₆, X₁through X₁, X₁ through X₈, X₁ through X₉, X₁ through X₁₀, X₁ throughX₁₁, and X₁ through X₁₂.

In various embodiments, X₁ is a thymidine, and/or X₂ is cytosine, and/orX₃ is a guanosine, and/or X₄ is a thymidine, and/or X₅ is a cytosine,and/or X₆ is a guanosine, and/or X₇ is a thymidine, and/or X₈ is athymidine, and/or X₉ is a thymidine, and/or X₁₀ is a thymidine, and/orX₁₁ is a thymidine, and/or X₁₂ is a cytosine. Those of ordinary skill inthe art will be able to determine the sequence of the remaining nucleicacids belonging to this family.

The nucleic acids of this family are generally at least 9 nucleotides inlength. In some embodiments, the nucleic acids are at least 10, at least12, at least 15, at least 18, at least 20, and at least 21 nucleotidesin length. In a preferred embodiment, the nucleic acids are 21nucleotides in length. In still further embodiments, the nucleic acidsare more than 21 nucleotides in length. Examples include nucleic acidsthat are at least 50, at least 75, at least 100, at least 200, at least500, at least 1000 nucleotides in length, or longer. Preferably, thenucleic acids are 9-100, and more preferably 21-100 nucleotides inlength.

All the nucleic acids of this first family contain at least one CpGmotif. These nucleic acids may contain two, three, four or more CpGmotifs. The CpG motifs may be contiguous to each other, oralternatively, they may be spaced apart from each other at constant orrandom distances.

The nucleic acids of this family also contain an overrepresentation ofthymidine nucleotides. These nucleic acids may contain at least 60%, atleast 55%, or at least 50% thymidines.

The invention is further premised, in part, on the unexpected discoveryof another family of nucleic acids that is as immunostimulatory aspreviously reported CpG nucleic acids. This family of nucleic acidscomprises the nucleotide sequence having the formula of5′ TCG TCG TTT TTC X₁X₂X₃ X₄X₅X₆ X₇X₈X₉ 3′ (SEQ ID NO:5)wherein X₁ through X₉ are independently selected residues that may beselected from the group of nucleotides consisting of adenosine,guanosine, thymidine, and cytosine. In some embodiments, there may be noflanking residues. As an example, the nucleic acid may comprise anucleotide sequence of 5′ TCG TCG TTT TTC 3′ (SEQ ID NO:6).

In other embodiments, the nucleic acid may lack X₉; X₉ and X₈; X₉, X₈and X₇; X₉ through X₆; X₉ through X₅; X₉ through X₄; X₉ through X₃; X₉through X₂; and X₉ through X₁.

In various embodiments, X₁ is a guanosine, and/or X₂ is guanosine,and/or X₃ is a thymidine, and/or X₄ is a cytosine, and/or X₅ is aguanosine, and/or X₆ is a thymidine, and/or X₇ is a thymidine, and/or X₈is a thymidine, and/or X₉ is a thymidine. Those of ordinary skill in theart will be able to determine the sequence of the remaining nucleicacids belonging to this family.

The nucleic acids of this family are generally at least 12 nucleotidesin length. In some embodiments, the nucleic acids are at least 15, atleast 18, and at least 21 nucleotides in length. In a preferredembodiment, the nucleic acids are 21 nucleotides in length. In stillfurther embodiments, the nucleic acids are more than 21 nucleotides inlength. Examples include nucleic acids that are at least 50, at least75, at least 100, at least 200, at least 500, at least 1000 nucleotidesin length, or longer. Preferably, the nucleic acids are 12-100, and morepreferably 21-100 nucleotides in length.

All the nucleic acids of this second family contain at least two CpGmotifs. These nucleic acids may contain three or four or more CpGmotifs, depending upon the embodiment. The CpG motifs may be contiguousto each other, or alternatively, they may be spaced apart from eachother at constant or random distances.

The nucleic acids of this family also contain an overrepresentation ofthymidine nucleotides. These nucleic acids may contain at least 60%, atleast 55%, or at least 50% thymidines.

In another aspect, the invention provides a nucleic acid comprising thenucleotide sequence of TCG TCG TTT TTC GGT CGT TTT (SEQ ID NO:1). Asdescribed in greater detail in the Examples, this nucleic acid wasidentified only after screening a multitude of nucleic acids for thosehaving similar or greater immunostimulatory activity than previouslyidentified immunostimulatory nucleic acids. More specifically, thenucleic acids were compared to a nucleic acid having a nucleotidesequence of TCG TCG TTT TGT CGT TTT GTC GTT (SEQ ID NO:2) that waspreviously shown to be immunostimulatory. The nucleic acid comprisingSEQ ID NO:1 was identified only after screening approximately 165nucleic acids for those having immunostimulatory capacity similar to orgreater than that of nucleic acids comprising SEQ ID NO:2. Thedifference in activity is surprising because there is only a minimaldifference between SEQ ID NO:1 and SEQ ID NO:2 (i.e., SEQ ID NO:1includes three additional internal nucleotides (i.e., TCG), and lackssix 3′ nucleotides as compared to SEQ ID NO:2). It was unexpected thatsuch a change in sequence would result in an increase inimmunostimulation.

In yet other aspects of the invention, nucleic acids having thefollowing nucleotide sequences are provided: 5′ TCG TCG TTT TTC GGT CGTTT 3′ (SEQ ID NO:7); 5′ TCG TCG TTT TTC GGT CGT T 3′ (SEQ ID NO:8); 5′TCG TCG TTT TTC GGT CGT 3′ (SEQ ID NO:9); 5′ TCG TCG TTT TTC GGT CG 3′(SEQ ID NO:10); 5′ TCG TCG TTT TTC GGT C 3′ (SEQ ID NO:11); 5′ TCG TCGTTT TTC GGT 3′ (SEQ ID NO:12); 5′ TCG TCG TTT TTC GG 3′ (SEQ ID NO:13);5′ TCG TCG TTT TTC G 3′ (SEQ ID NO:27); 5′ TCG TCG TTT TTC 3′ (SEQ IDNO:14); 5′ TCG TCG TTT TTC GGT CGT TTT 3′ (SEQ ID NO:15), 5′ CG TCG TTTTTC GGT CGT TTT 3′ (SEQ ID NO:16), 5′ G TCG TTT TTC GGT CGT TTT 3′ (SEQID NO:17), 5′ TCG TTT TTC GGT CGT TTT 3′ (SEQ ID NO:18), 5′ CG TTT TTCGGT CGT TTT 3′ (SEQ ID NO:19), 5′ G TTT TTC GGT CGT TTT 3′ (SEQ IDNO:20), 5′ TTT TTC GGT CGT TTT 3′ (SEQ ID NO:21), 5′ TT TTC GGT CGT TTT3′ (SEQ ID NO:22), 5′ T TTC GGT CGT TTT 3′ (SEQ ID NO:23), 5′ TTC GGTCGT TTT 3′ (SEQ ID NO:24), 5′ TC GGT CGT TTT 3′ (SEQ ID NO:25), 5′ C GGTCGT TTT 3′ (SEQ ID NO:26), 5′ GGT CGT TTT 3′ (SEQ ID NO:4).

These immunostimulatory nucleic acids are capable of activating theinnate immune system, and augmenting both humoral and cellular antigenspecific responses when co-administered with an antigen, such asHepatitis B surface antigen. The Examples provided herein demonstratethat these nucleic acids can stimulate human immune cells in vitro, andmurine cells in vitro and in vivo. When compared to a sequence known tobe a potent adjuvant, the nucleic acid of SEQ ID NO:1 is at least 10-15%as a vaccine adjuvant.

The CpG motifs of the nucleic acids described herein are preferablyunmethylated. An unmethylated CpG motif is an unmethylatedcytosine-guanine dinucleotide sequence (i.e. an unmethylated 5′ cytosinefollowed by 3′ guanosine and linked by a phosphate bond). All thenucleic acid described herein are immunostimulatory. In some embodimentsof the invention, the CpG motifs are methylated. A methylated CpG motifis a methylated cytosine-guanine dinucleotide sequence (i.e., amethylated 5′ cytosine followed by a 3′ guanosine and linked by aphosphate bond).

A CpG nucleic acid is a nucleic acid that comprises the formula5′ X₁X₂CGX₃X₄ 3′wherein C is unmethylated, wherein X₁X₂ and X₃X₄ are nucleotides. In arelated embodiment, the 5′ X₁X₂CGX₃ X₄ 3′ sequence is a non-palindromicsequence. In certain embodiments, X₁X₂ are nucleotides selected from thegroup consisting of GpT, GpG, GpA, ApA, ApT, ApG, CpT, CpA, CpG, TpA,TpT, and TpG; and X₃X₄ are nucleotides selected from the groupconsisting of TpT, CpT, ApT, TpG, ApG, CpG, TpC, ApC, CpC, TpA, ApA, andCpA. In more particular embodiments, X₁X₂ are nucleotides selected fromthe group consisting of GpA and GpT; and X₃X₄ are TpT. In yet otherembodiments, X₁X₂ are both purines and X₃X₄ are both pyrimidines. Inanother embodiment, X₂ is a T and X₃ is a pyrimidine. Examples of CpGnucleic acids are described in U.S. Non-Provisional patent applicationSer. No. 09/669,187, filed Sep. 25, 2000, and in published PCT PatentApplication PCT/US00/26383, having publication number WO01/22972.

The nucleic acids of the invention can further contain otherimmunostimulatory motifs such as poly T motifs, poly G motifs, TGmotifs, poly A motifs, poly C motifs, and the like, provided that thecore sequences of SEQ ID NO:4 and SEQ ID NO:6 are present. Theseimmunostimulatory motifs are described in greater detail below or inU.S. Non-Provisional patent application Ser. No. 09/669,187, filed Sep.25, 2000, and published PCT Patent Application PCT/US00/26383, havingpublication number WO01/22972

A T-rich nucleic acid is a nucleic acid which includes at least one polyT sequence and/or which has a nucleotide composition of greater than 25%T nucleotide residues. A nucleic acid having a poly-T sequence includesat least four Ts in a row, such as 5′TTTT3′. Preferably a T-rich nucleicacid includes more than one poly T sequence. In preferred embodimentsthe T-rich nucleic acid may have 2, 3, 4, etc poly T sequences. OtherT-rich nucleic acids according to the invention have a nucleotidecomposition of greater than 25% T nucleotide residues, but do notnecessarily include a poly T sequence. In these T-rich nucleic acids theT nucleotide resides may be separated from one another by other types ofnucleotide residues, i.e., G, C, and A. In some embodiments the T-richnucleic acids have a nucleotide composition of greater than 35%, 40%,50%, 60%, 70%, 80%, 90%, and 99%, T nucleotide residues and everyinteger % in between. Preferably the T-rich nucleic acids have at leastone poly T sequence and a nucleotide composition of greater than 25% Tnucleotide residues.

Poly G nucleic acids preferably are nucleic acids having the followingformulas:5′ X₁X₂GGGX₃X₄ 3′wherein X_(1,) X_(2,) X_(3,) and X₄ are nucleotides. In preferredembodiments at least one of X₃ and X₄ are a G. In other embodiments bothof X₃ and X₄ are a G. In yet other embodiments the preferred formula is5′ GGGNGGG 3′ (SEQ ID NO:28) or 5′ GGGNGGGNGGG 3′ (SEQ ID NO:29),wherein N represents between 0 and 20 nucleotides.

A C-rich nucleic acid is a nucleic acid molecule having at least one orpreferably at least two poly-C regions or which is composed of at least50% C nucleotides. A poly-C region is at least four C residues in a row.Thus a poly-C region is encompassed by the formula 5′CCCC 3′. In someembodiments it is preferred that the poly-C region have the formula5′CCCCCC 3′. Other C-rich nucleic acids according to the invention havea nucleotide composition of greater than 50% C nucleotide residues, butdo not necessarily include a poly C sequence. In these C-rich nucleicacids the C nucleotide residues may be separated from one another byother types of nucleotide residues, i.e., G, T, and A. In someembodiments the C-rich nucleic acids have a nucleotide composition ofgreater than 60%, 70%, 80%, 90%, and 99%, C nucleotide residues andevery integer % in between. Preferably the C-rich nucleic acids have atleast one poly C sequence and a nucleotide composition of greater than50% C nucleotide residues, and in some embodiments are also T-rich.

The immunostimulatory nucleic acids can be double-stranded orsingle-stranded. Generally, double-stranded molecules are more stable invivo, while single-stranded molecules have increased immune activity.Thus in some aspects of the invention it is preferred that the nucleicacid be single stranded and in other aspects it is preferred that thenucleic acid be double stranded.

The terms “nucleic acid” and “oligonucleotide” are used interchangeablyherein to mean multiple nucleotides (i.e. molecules comprising a sugar(e.g. ribose or deoxyribose) linked to a phosphate group and to anexchangeable organic base, which is either a substituted pyrimidine(e.g. cytosine (C), thymidine (T) or uracil (U)) or a substituted purine(e.g. adenine (A) or guanine (G)). As used herein, the terms refer tooligoribonucleotides as well as oligodeoxyribonucleotides. The termsshall also include polynucleosides (i.e. a polynucleotide minus thephosphate) and any other organic base containing polymer. Nucleic acidmolecules can be obtained from existing nucleic acid sources (e.g.,genomic or cDNA), but are preferably synthetic (e.g. produced by nucleicacid synthesis).

The immunostimulatory oligonucleotides of the instant invention canencompass various chemical modifications and substitutions, incomparison to natural RNA and DNA, involving a phosphodiesterinternucleoside bridge, a β-D-ribose unit and/or a natural nucleosidebase (adenine, guanine, cytosine, thymine, uracil). Examples of chemicalmodifications are known to the skilled person and are described, forexample, in Uhlmann E et al. (1990) Chem Rev 90:543; “Protocols forOligonucleotides and Analogs” Synthesis and Properties & Synthesis andAnalytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993;Crooke S T et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; andHunziker J et al. (1995) Mod Synth Methods 7:331-417. An oligonucleotideaccording to the invention may have one or more modifications, whereineach modification is located at a particular phosphodiesterinternucleoside bridge and/or at a particular β-D-ribose unit and/or ata particular natural nucleoside base position in comparison to anoligonucleotide of the same sequence which is composed of natural DNA orRNA.

For example, the oligonucleotides may comprise one or more modificationsand wherein each modification is independently selected from:

-   a) the replacement of a phosphodiester internucleoside bridge    located at the 3′ and/or the 5′ end of a nucleoside by a modified    internucleoside bridge,-   b) the replacement of phosphodiester bridge located at the 3′ and/or    the 5′ end of a nucleoside by a dephospho bridge,-   c) the replacement of a sugar phosphate unit from the sugar    phosphate backbone by another unit,-   d) the replacement of a β-D-ribose unit by a modified sugar unit,    and-   e) the replacement of a natural nucleoside base by a modified    nucleoside base.

More detailed examples for the chemical modification of anoligonucleotide are as follows.

Nucleic acids also include substituted purines and pyrimidines such asC-5 propyne pyrimidine and 7-deaza-7-substituted purine modified bases.Wagner R W et al. (1996) Nat Biotechnol 14:840-4. Purines andpyrimidines include but are not limited to adenine, cytosine, guanine,thymidine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine,2,6-diaminopurine, hypoxanthine, and other naturally and non-naturallyoccurring nucleobases, substituted and unsubstituted aromatic moieties.Other such modifications are well known to those of skill in the art. Inall of the foregoing embodiments, an X residue can also be anon-naturally occurring nucleotide, or a nucleotide analog, such asthose described herein.

A modified base is any base which is chemically distinct from thenaturally occurring bases typically found in DNA and RNA such as T, C,G, A, and U, but which share basic chemical structures with thesenaturally occurring bases. The modified nucleoside base may be, forexample, selected from hypoxanthine, uracil, dihydrouracil,pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil,5-(C₁-C₆)-alkyluracil, 5-(C₂-C₆)-alkenyluracil, 5-(C₂-C₆)-alkynyluracil,5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil,5-hydroxycytosine, 5-(C₁-C₆)-alkylcytosine, 5-(C₂-C₆)-alkenylcytosine,5-(C₂-C₆)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine,5-bromocytosine, N²-dimethylguanine, 2,4-diamino-purine, 8-azapurine, asubstituted 7-deazapurine, preferably 7-deaza-7-substituted and/or7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N4-alkylcytosine,e.g., N4-ethylcytosine, 5-hydroxydeoxycytidine,5-hydroxymethyldeoxycytidine, N4-alkyldeoxycytidine, e.g.,N4-ethyldeoxycytidine, 6-thiodeoxyguanosine, and deoxyribonucleosides ofnitropyrrole, C5-propynylpyrimidine, and diaminopurine e.g.,2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine,2-amino-6-chloropurine, hypoxanthine or other modifications of a naturalnucleoside bases. This list is meant to be exemplary and is not to beinterpreted to be limiting.

In particular formulas described herein a set of modified bases isdefined. For instance the letter Y is used to refer to a nucleotidecontaining a cytosine or a modified cytosine. A modified cytosine asused herein is a naturally occurring or non-naturally occurringpyrimidine base analog of cytosine which can replace this base withoutimpairing the immunostimulatory activity of the oligonucleotide.Modified cytosines include but are not limited to 5-substitutedcytosines (e.g. 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine,5-bromo-cytosine, 5-iodo-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, 5-difluoromethyl-cytosine, and unsubstitutedor substituted 5-alkynyl-cytosine), 6-substituted cytosines,N4-substituted cytosines (e.g. N4-ethyl-cytosine), 5-aza-cytosine,2-mercapto-cytosine, isocytosine, pseudo-isocytosine, cytosine analogswith condensed ring systems (e.g. N,N′-propylene cytosine orphenoxazine), and uracil and its derivatives (e.g. 5-fluoro-uracil,5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil,5-propynyl-uracil). Some of the preferred cytosines include5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine,5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In another embodimentof the invention, the cytosine base is substituted by a universal base(e.g. 3-nitropyrrole, P-base), an aromatic ring system (e.g.fluorobenzene or difluorobenzene) or a hydrogen atom (dSpacer). Theletter Z is used to refer to guanine or a modified guanine base. Amodified guanine as used herein is a naturally occurring ornon-naturally occurring purine base analog of guanine which can replacethis base without impairing the immunostimulatory activity of theoligonucleotide. Modified guanines include but are not limited to7-deazaguanine, 7-deaza-7-substituted guanine (such as7-deaza-7-(C2-C6)alkynylguanine), 7-deaza-8-substituted guanine,hypoxanthine, N2-substituted guanines (e.g. N2-methyl-guanine),5-amino-3-methyl-3H,6H-thiazolo[4,5-d]pyrimidine-2,7-dione,2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substitutedadenines (e.g. N6-methyl-adenine, 8-oxo-adenine) 8-substituted guanine(e.g. 8-hydroxyguanine and 8-bromoguanine), and 6-thioguanine. Inanother embodiment of the invention, the guanine base is substituted bya universal base (e.g. 4-methyl-indole, 5-nitro-indole, and K-base), anaromatic ring system (e.g. benzimidazole or dichloro-benzimidazole,1-methyl-1H-[1,2,4]triazole-3-carboxylic acid amide) or a hydrogen atom(dSpacer).

The oligonucleotides may include modified internucleotide linkages, suchas those described in a or b above. These modified linkages may bepartially resistant to degradation (e.g., are stabilized). A “stabilizednucleic acid molecule” shall mean a nucleic acid molecule that isrelatively resistant to in vivo degradation (e.g. via an exo- orendo-nuclease). Stabilization can be a function of length or secondarystructure. Nucleic acids that are tens to hundreds of kilobases long arerelatively resistant to in vivo degradation. For shorter nucleic acids,secondary structure can stabilize and increase their effect. Forexample, if the 3′ end of an nucleic acid has self-complementarity to anupstream region, so that it can fold back and form a sort of stem loopstructure, then the nucleic acid becomes stabilized and thereforeexhibits more activity.

Nucleic acid stabilization can also be accomplished via phosphatebackbone modifications. Oligonucleotides having phosphorothioatelinkages, in some embodiments, may provide maximal activity and protectthe oligonucleotide from degradation by intracellular exo- andendo-nucleases.

It has been demonstrated that modification of the nucleic acid backboneprovides enhanced activity of nucleic acids when administered in vivo.Constructs having phosphorothioate linkages provide maximal activity andprotect the nucleic acid from degradation by intracellular exo- andendo-nucleases. Other modified nucleic acids include phosphodiestermodified nucleic acids, combinations of phosphodiester andphosphorothioate nucleic acid, methylphosphonate,methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinationsthereof. Each of these combinations and their particular effects onimmune cells is discussed in more detail with respect to CpG nucleicacids in PCT Published Patent Applications PCT/US95/01570 (WO 96/02555)and PCT/US97/19791 (WO 98/18810) and in U.S. patents U.S. Pat. No.6,194,388 B1 issued Feb. 27, 2001 and U.S. Pat. No. 6,239,116 B1 issuedMay 29, 2001, the entire contents of which are hereby incorporated byreference. It is believed that these modified nucleic acids may showmore stimulatory activity due to enhanced nuclease resistance, increasedcellular uptake, increased protein binding, and/or altered intracellularlocalization.

Other stabilized nucleic acids include: nonionic DNA analogs, such asalkyl- and aryl-phosphates (in which the charged phosphonate oxygen isreplaced by an alkyl or aryl group), phosphodiester andalkylphosphotriesters, in which the charged oxygen moiety is alkylated.Nucleic acids which contain diol, such as tetraethyleneglycol orhexaethyleneglycol, at either or both termini have also been shown to besubstantially resistant to nuclease degradation.

The oligonucleotides may have one or two accessible 5′ ends. It ispossible to create modified oligonucleotides having two such 5′ ends,for instance, by attaching two oligonucleotides through a 3′-3′ linkageto generate an oligonucleotide having one or two accessible 5′ ends. The3′3′-linkage may be a phosphodiester, phosphorothioate or any othermodified internucleoside bridge. Methods for accomplishing such linkagesare known in the art. For instance, such linkages have been described inSeliger, H. et al., Oligonucleotide analogs with terminal 3′-3′- and5′-5′-internucleotidic linkages as antisense inhibitors of viral geneexpression, Nucleosides & Nucleotides (1991), 10(1-3), 469-77 and Jiang,et al., Pseudo-cyclic oligonucleotides: in vitro and in vivo properties,Bioorganic & Medicinal Chemistry (1999), 7(12), 2727-2735.

Additionally, 3′3′-linked ODNs where the linkage between the 3′-terminalnucleosides is not a phosphodiester, phosphorothioate or other modifiedbridge, can be prepared using an additional spacer, such as tri- ortetra-ethylenglycol phosphate moiety (Durand, M. et al, Triple-helixformation by an oligonucleotide containing one (dA)12 and two (dT)12sequences bridged by two hexaethylene glycol chains, Biochemistry(1992), 31(38), 9197-204, U.S. Pat. Nos. 5,658,738, and 5,668,265).Alternatively, the non-nucleotidic linker may be derived fromethanediol, propanediol, or from an abasic deoxyribose (dSpacer) unit(Fontanel, Marie Laurence et al., Sterical recognition by T4polynucleotide kinase of non-nucleosidic moieties 5′-attached tooligonucleotides; Nucleic Acids Research (1994), 22(11), 2022-7) usingstandard phosphoramidite chemistry. The non-nucleotidic linkers can beincorporated once or multiple times, or combined with each otherallowing for any desirable distance between the 3′-ends of the two ODNsto be linked.

A phosphodiester internucleoside bridge located at the 3′ and/or the 5′end of a nucleoside can be replaced by a modified internucleosidebridge, wherein the modified internucleoside bridge is for exampleselected from phosphorothioate, phosphorodithioate,NR¹R²-phosphoramidate, boranophosphate, α-hydroxybenzyl phosphonate,phosphate-(C₁-C₂₁)—O-alkyl ester,phosphate-[(C₆-C₁₂)aryl-(C₁-C₂₁)—O-alkyl]ester, (C₁-C₈)alkylphosphonateand/or (C₆-C₁₂)arylphosphonate bridges, (C₇-C₁₂)-α-hydroxymethyl-aryl(e.g., disclosed in WO 95/01363), wherein (C₆-C₁₂)aryl, (C₆-C₂₀)aryl and(C₆-C₁₄)aryl are optionally substituted by halogen, alkyl, alkoxy,nitro, cyano, and where R¹ and R² are, independently of each other,hydrogen, (C₁-C₁₈)-alkyl, (C₆-C₂₀)-aryl, (C₆-C₁₄)-aryl-(C₁-C₈)-alkyl,preferably hydrogen, (C₁-C₈)-alkyl, preferably (C₁-C₄)-alkyl and/ormethoxyethyl, or R¹ and R² form, together with the nitrogen atomcarrying them, a 5-6-membered heterocyclic ring which can additionallycontain a further heteroatom from the group O, S and N.

The replacement of a phosphodiester bridge located at the 3′ and/or the5′ end of a nucleoside by a dephospho bridge (dephospho bridges aredescribed, for example, in Uhlmann E and Peyman A in “Methods inMolecular Biology”, Vol. 20, “Protocols for Oligonucleotides andAnalogs”, S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp.355 ff), wherein a dephospho bridge is for example selected from thedephospho bridges formacetal, 3′-thioformacetal, methylhydroxylamine,oxime, methylenedimethyl-hydrazo, dimethylenesulfone and/or silylgroups.

The compositions of the invention may optionally be have chimericbackbones. As used herein, a chimeric backbone is one that comprisesmore than one type of linkage. In one embodiment, the chimeric backbonecan be represented by the formula: 5′ Y₁N₁ZN₂Y₂ 3′. Y₁ and Y₂ arenucleic acid molecules having between 1 and 10 nucleotides. Y₁ and Y₂each include at least one modified internucleotide linkage. Since atleast 2 nucleotides of the chimeric oligonucleotides include backbonemodifications these nucleic acids are an example of one type of“stabilized immunostimulatory nucleic acids.”

With respect to the chimeric oligonucleotides, Y₁ and Y₂ are consideredindependent of one another. This means that each of Y₁ and Y₂ may or maynot have different sequences and different backbone linkages from oneanther in the same molecule. In some embodiments Y₁ and/or Y₂ havebetween 3 and 8 nucleotides. N₁ and N₂ are nucleic acid molecules havingbetween 0 and 5 nucleotides as long as N₁ZN₂ has at least 6 nucleotidesin total. The nucleotides of N₁ZN₂ have a phosphodiester backbone and donot include nucleic acids having a modified backbone. Z is animmunostimulatory nucleic acid motif, preferably selected from thoserecited herein.

The center nucleotides (N₁ZN₂) of the formula Y₁N₁ZN₂Y₂ havephosphodiester internucleotide linkages and Y₁ and Y₂ have at least one,but may have more than one or even may have all modified internucleotidelinkages. In preferred embodiments Y₁ and/or Y₂ have at least two orbetween two and five modified internucleotide linkages or Y₁ has twomodified internucleotide linkages and Y₂ has five modifiedinternucleotide linkages or Y₁ has five modified internucleotidelinkages and Y₂ has two modified internucleotide linkages. The modifiedinternucleotide linkage, in some embodiments is a phosphorothioatemodified linkage, a phosphorodithioate modified linkage or a p-ethoxymodified linkage.

The nucleic acids also include nucleic acids having backbone sugarswhich are covalently attached to low molecular weight organic groupsother than a hydroxyl group at the 2′ position and other than aphosphate group at the 5′ position. Thus, modified nucleic acids mayinclude a 2′-O-alkylated ribose group. In addition, modified nucleicacids may include sugars such as arabinose or 2′-fluoroarabinose insteadof ribose. Thus the nucleic acids may be heterogeneous in backbonecomposition thereby containing any possible combination of polymer unitslinked together such as peptide-nucleic acids (which have amino acidbackbone with nucleic acid bases). In some embodiments, the nucleicacids are homogeneous in backbone composition. Other examples aredescribed in more detail below.

A sugar phosphate unit (i.e., a β-D-ribose and phosphodiesterinternucleoside bridge together forming a sugar phosphate unit) from thesugar phosphate backbone (i.e., a sugar phosphate backbone is composedof sugar phosphate units) can be replaced by another unit, wherein theother unit is for example suitable to build up a “morpholino-derivative”oligomer (as described, for example, in Stirchak E P et al. (1989)Nucleic Acids Res 17:6129-41), that is, e.g., the replacement by amorpholino-derivative unit; or to build up a polyamide nucleic acid(“PNA”; as described for example, in Nielsen PE et al. (1994) BioconjugChem 5:3-7), that is, e.g., the replacement by a PNA backbone unit,e.g., by 2-aminoethylglycine. The oligonucleotide may have othercarbohydrate backbone modifications and replacements, such as peptidenucleic acids with phosphate groups (PHONA), locked nucleic acids (LNA),and oligonucleotides having backbone sections with alkyl linkers oramino linkers. The alkyl linker may be branched or unbranched,substituted or unsubstituted, and chirally pure or a racemic mixture.

A β-ribose unit or a β-D-2′-deoxyribose unit can be replaced by amodified sugar unit, wherein the modified sugar unit is for exampleselected from β-D-ribose, α-D-2′-deoxyribose, L-2′-deoxyribose,2′-F-2′-deoxyribose, 2′-F-arabinose, 2′-O—(C₁-C₆)alkyl-ribose,preferably 2′-O—(C₁-C₆)alkyl-ribose is 2′-O-methylribose,2′-O—(C₂-C₆)alkenyl-ribose, 2′-[O—(C₁-C₆)alkyl-O—(C₁-C₆)alkyl]-ribose,2′-NH₂-2′-deoxyribose, β-D-xylo-furanose, α-arabinofuranose,2,4-dideoxy-β-D-erythro-hexo-pyranose, and carbocyclic (described, forexample, in Froehler J (1992) Am Chem Soc 114:8320) and/or open-chainsugar analogs (described, for example, in Vandendriessche et al. (1993)Tetrahedron 49:7223) and/or bicyclosugar analogs (described, forexample, in Tarkov M et al. (1993) Helv Chim Acta 76:481).

In some embodiments the sugar is 2′-O-methylribose, particularly for oneor both nucleotides linked by a phosphodiester or phosphodiester-likeinternucleoside linkage.

For use in the instant invention, the oligonucleotides of the inventioncan be synthesized de novo using any of a number of procedures wellknown in the art. For example, the b-cyanoethyl phosphoramidite method(Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859, 1981);nucleoside H-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054,1986; Froehler et al., Nucl. Acid. Res. 14:5399-5407, 1986; Garegg etal., Tet. Let. 27:4055-4058, 1986, Gaffney et al., Tet. Let.29:2619-2622, 1988). These chemistries can be performed by a variety ofautomated nucleic acid synthesizers available in the market. Theseoligonucleotides are referred to as synthetic oligonucleotides.Alternatively, T-rich and/or TG dinucleotides can be produced on a largescale in plasmids, (see Sambrook, T., et al., “Molecular Cloning: ALaboratory Manual”, Cold Spring Harbor laboratory Press, New York, 1989)and separated into smaller pieces or administered whole. Nucleic acidscan be prepared from existing nucleic acid sequences (e.g., genomic orcDNA) using known techniques, such as those employing restrictionenzymes, exonucleases or endonucleases.

Modified backbones such as phosphorothioates may be synthesized usingautomated techniques employing either phosphoramidate or H-phosphonatechemistries. Aryl-and alkyl-phosphonates can be made, e.g., as describedin U.S. Pat. No. 4,469,863; and alkylphosphotriesters (in which thecharged oxygen moiety is alkylated as described in U.S. Pat. No.5,023,243 and European Patent No. 092,574) can be prepared by automatedsolid phase synthesis using commercially available reagents. Methods formaking other DNA backbone modifications and substitutions have beendescribed (e.g., Uhlmann, E. and Peyman, A., Chem. Rev. 90:544, 1990;Goodchild, J., Bioconjugate Chem. 1:165, 1990).

Nucleic acids prepared in this manner are referred to as isolatednucleic acid. An “isolated nucleic acid” generally refers to a nucleicacid which is separated from components with which it is normallyassociated in nature. As an example, an isolated nucleic acid may be onewhich is separated from a cell, from a nucleus, from mitochondria orfrom chromatin.

In the case where the nucleic acid is administered in conjunction withan antigen that is encoded in a nucleic acid vector (as describedherein), it is preferred that the backbone of the nucleic acid be achimeric combination of phosphodiester and phosphorothioate (or otherphosphate modification). The cell may have a problem taking up a plasmidvector in the presence of completely phosphorothioate nucleic acid. Thuswhen both a vector and a nucleic acid are delivered to a subject, it ispreferred that the nucleic acid have a chimeric backbone or have aphosphorothioate backbone but that the plasmid be associated with avehicle that delivers it directly into the cell, thus avoiding the needfor cellular uptake. Such vehicles are known in the art and include, forexample, liposomes and gene guns.

The invention further embraces the use of any of these foregoing nucleicacids in the methods recited herein, as well as all previously describedand previously known uses of immunostimulatory nucleic acids.

It has been discovered according to the invention that theimmunostimulatory nucleic acids have surprisingly increased immunestimulatory effects. For example, it has been demonstrated that thenucleic acids described herein are able to provide protection againstinfection, probably by generally stimulating the immune system. TheExamples illustrate the ability of the nucleic acid having a nucleotidesequence of SEQ ID NO: 1 to protect murine subjects challenged withHerpes Simplex Virus 2 (HSV-2). The nucleic acid can administered priorto or at the same time as viral challenge.

The demonstrated ability of these nucleic acids to induce immunestimulation is evidence that the nucleic acids are effective therapeuticagents for vaccination, cancer immunotherapy, asthma immunotherapy,general enhancement of immune function, enhancement of hematopoieticrecovery following radiation or chemotherapy, and other immunemodulatory applications in humans and other subjects.

The nucleic acids of the invention can be used as stand alone therapies.A stand alone therapy is a therapy in which a prophylactically ortherapeutically beneficial result can be achieved from theadministration of a single agent or composition. Accordingly, thenucleic acids disclosed herein can be used alone in the prevention ortreatment of infectious disease, cancer, and asthma and allergy, becausethe nucleic acids are capable of inducing immune responses that arebeneficial to the therapeutic outcome of these diseases. Some of themethods described herein relate to the use of nucleic acids as a standalone therapy, while others related to the use of the nucleic acids incombination with other therapeutic agents.

When used in a vaccine, the nucleic acid is administered with anantigen. Preferably, the antigen is specific for the disorder sought tobe prevented or treated. For example, if the disorder is an infectiousdisease, the antigen is preferably derived from the infectious organism(e.g., bacterium, virus, parasite, fungus, etc.). If the disorder is acancer, the antigen is preferably a cancer antigen.

The immunostimulatory nucleic acids are useful in some aspects of theinvention as a prophylactic vaccine for the prevention of an infection(i.e., an infectious disease), a cancer, an allergy, or asthma.Preferably, prophylactic vaccination is used in subjects that are notdiagnosed with one of these conditions, and more preferably the subjectsare considered at risk of developing one of these conditions. Forexample, the subject may be one that is at risk of developing aninfection with an infectious organism, or one that is at risk ofdeveloping a cancer in which a specific cancer antigen has beenidentified, or one that is at risk of developing an allergy for which anallergen is known, or one that is at risk of developing asthma where thepredisposition to asthma is known.

A subject at risk, as used herein, is a subject who has any risk ofexposure to an infection causing pathogen, a carcinogen, or an allergen.A subject at risk also includes subjects that have a predisposition todeveloping such disorders. Some predispositions can be genetic (and canthereby be identified either by genetic analysis or by family history).Some predispositions are environmental (e.g., prior exposure tocarcinogens, etc.) An example of a subject at risk of developing aninfection is a subject living in or expecting to travel to an area wherea particular type of infectious agent is or has been found, or it may bea subject who through lifestyle or medical procedures is exposed to anorganism either directly or indirectly by contact with bodily fluidsthat may contain infectious organisms. Subjects at risk of developinginfection also include general populations to which a medical agencyrecommends vaccination for a particular infectious organism.

If the antigen is an allergen and the subject develops allergicresponses to that particular antigen and the subject may be exposed tothe antigen, i.e., during pollen season, then that subject is at risk ofexposure to the antigen. A subject at risk of developing an allergy toasthma includes those subjects that have been identified as having anallergy or asthma but that don't have the active disease during theimmunostimulatory nucleic acid treatment as well as subjects that areconsidered to be at risk of developing these diseases because of geneticor environmental factors.

The immunostimulatory nucleic acids can also be given without theantigen or allergen for shorter term protection against infection,allergy or cancer, and in this case repeated doses will allow longerterm protection.

A subject at risk of developing a cancer is one who is who has a highprobability of developing cancer (e.g., a probability that is greaterthan the probability within the general public). These subjects include,for instance, subjects having a genetic abnormality, the presence ofwhich has been demonstrated to have a correlative relation to alikelihood of developing a cancer that is greater than the likelihood ofthe general public, and subjects exposed to cancer causing agents (i.e.,carcinogens) such as tobacco, asbestos, or other chemical toxins, or asubject who has previously been treated for cancer and is in apparentremission. When a subject at risk of developing a cancer is treated withan antigen specific for the type of cancer to which the subject is atrisk of developing and a immunostimulatory nucleic acid, the subject maybe able to kill the cancer cells as they develop. If a tumor begins toform in the subject, the subject will develop a specific immune responseagainst the tumor antigen.

In addition to the use of the immunostimulatory nucleic acids as aprophylactic, the invention also encompasses the use of theimmunostimulatory nucleic acids for the treatment of a subject having aninfection, an allergy, asthma, or a cancer.

A subject having an infection is a subject that has been exposed to aninfectious pathogen and has acute or chronic detectable levels of thepathogen in the body, or in bodily waste. When used therapeutically, theimmunostimulatory nucleic acids can be used as a stand alone or incombination with another therapeutic agent. For example, theimmunostimulatory nucleic acids can be used therapeutically with anantigen to mount an antigen specific systemic or mucosal immune responsethat is capable of reducing the level of, or eradicating, the infectiouspathogen.

An infectious disease, as used herein, is a disease arising from thepresence of a foreign microorganism in the body. It is particularlyimportant to develop effective vaccine strategies and treatments toprotect the body's mucosal surfaces, which are the primary site ofpathogenic entry.

As used herein, the term treat, treated, or treating when used withrespect to an infectious disease refers to a prophylactic treatmentwhich increases the resistance of a subject (a subject at risk ofinfection) to infection with a pathogen or, in other words, decreasesthe likelihood that the subject will become infected with the pathogenas well as a treatment after the subject (a subject who has beeninfected) has become infected in order to fight the infection, e.g.,reduce or eliminate the infection or prevent it from becoming worse.

A subject having an allergy is a subject that has or is at risk ofdeveloping an allergic reaction in response to an allergen. An allergyrefers to acquired hypersensitivity to a substance (allergen). Allergicconditions include but are not limited to eczema, allergic rhinitis orcoryza, hay fever, conjunctivitis, bronchial asthma, urticaria (hives)and food allergies, and other atopic conditions.

Currently, allergic diseases are generally treated by the injection ofsmall doses of antigen followed by subsequent increasing dosage ofantigen. It is believed that this procedure induces tolerization to theallergen to prevent further allergic reactions. These methods, however,can take several years to be effective and are associated with the riskof side effects such as anaphylactic shock. The methods of the inventionavoid these problems.

Allergies are generally caused by IgE antibody generation againstharmless allergens. The cytokines that are induced by systemic ormucosal administration of immunostimulatory nucleic acids arepredominantly of a class called Th1 (examples are IL-12 and IFN-γ) andthese induce both humoral and cellular immune responses. The types ofantibodies associated with a Th1 response are generally more protectivebecause they have high neutralization and opsonization capabilities. Theother major type of immune response, which is associated with theproduction of IL-4, IL-5 and IL-10 cytokines, is termed a Th2 immuneresponse. Th2 responses involve predominately antibodies and these haveless protective effect against infection and some Th2 isotypes (e.g.,IgE) are associated with allergy. In general, it appears that allergicdiseases are mediated by Th2 type immune responses while Th1 responsesprovide the best protection against infection, although excessive Th1responses are associated with autoimmune disease. Based on the abilityof the immunostimulatory nucleic acids to shift the immune response in asubject from a Th2 (which is associated with production of IgEantibodies and allergy) to a Th1 response (which is protective againstallergic reactions), an effective dose for inducing an immune responseof a immunostimulatory nucleic acid can be administered to a subject totreat or prevent an allergy.

Thus, the immunostimulatory nucleic acids have significant therapeuticutility in the treatment of allergic and non-allergic conditions such asasthma. Th2 cytokines, especially IL-4 and IL-5 are elevated in theairways of asthmatic subjects. These cytokines promote important aspectsof the asthmatic inflammatory response, including IgE isotope switching,eosinophil chemotaxis and activation and mast cell growth. Th1cytokines, especially IFN-γ and IL-12, can suppress the formation of Th2clones and production of Th2 cytokines. Asthma refers to a disorder ofthe respiratory system characterized by inflammation, narrowing of theairways and increased reactivity of the airways to inhaled agents.Asthma is frequently, although not exclusively associated with atopic orallergic symptoms.

A subject having a cancer is a subject that has detectable cancerouscells. The cancer may be a malignant or non-malignant cancer. Cancers ortumors include but are not limited to biliary tract cancer; braincancer; breast cancer; cervical cancer; choriocarcinoma; colon cancer;endometrial cancer; esophageal cancer; gastric cancer; intraepithelialneoplasms; lymphomas; liver cancer; lung cancer (e.g. small cell andnon-small cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;pancreas cancer; prostate cancer; rectal cancer; sarcomas; skin cancer;testicular cancer; thyroid cancer; and renal cancer, as well as othercarcinomas and sarcomas. In one embodiment the cancer is hairy cellleukemia, chronic myelogenous leukemia, cutaneous T-cell leukemia,multiple myeloma, follicular lymphoma, malignant melanoma, squamous cellcarcinoma, renal cell carcinoma, prostate carcinoma, bladder cellcarcinoma, or colon carcinoma.

Some cancer cells are antigenic and thus can be targeted by the immunesystem. In one aspect, the combined administration of immunostimulatorynucleic acids and cancer medicaments, particularly those which areclassified as cancer immunotherapies, is useful for stimulating aspecific immune response against a cancer antigen.

The theory of immune surveillance is that a prime function of the immunesystem is to detect and eliminate neoplastic cells before a tumor forms.A basic principle of this theory is that cancer cells are antigenicallydifferent from normal cells and thus elicit immune reactions that aresimilar to those that cause rejection of immunologically incompatibleallografts. Studies have confirmed that tumor cells differ, eitherqualitatively or quantitatively, in their expression of antigens. Suchantigens are referred to interchangeably as tumor antigens or cancerantigens. Some of these antigens may in turn be tumor-specific antigensor tumor-associated antigens. “Tumor-specific antigens” are antigensthat are specifically present in tumor cells but not normal cells.Examples of tumor specific antigens are viral antigens in tumors inducedby DNA or RNA viruses. “Tumor-associated” antigens are present in bothtumor cells and normal cells but are present in a different quantity ora different form in tumor cells. Examples of such antigens are oncofetalantigens (e.g., carcinoembryonic antigen), differentiation antigens(e.g., T and Tn antigens), and oncogene products (e.g., HER/neu).

Different types of cells that can kill tumor targets in vitro and invivo have been identified: natural killer cells (NK cells), cytolytic Tlymphocytes (CTLs), lymphokine-activated killer cells (LAKs), andactivated macrophages. NK cells can kill tumor cells without having beenpreviously sensitized to specific antigens, and the activity does notrequire the presence of class I antigens encoded by the majorhistocompatibility complex (MHC) on target cells. NK cells are thoughtto participate in the control of nascent tumors and in the control ofmetastatic growth. In contrast to NK cells, CTLs can kill tumor cellsonly after they have been sensitized to tumor antigens and when thetarget antigen is expressed on the tumor cells that also express MHCclass I. CTLs are thought to be effector cells in the rejection oftransplanted tumors and of tumors caused by DNA viruses. LAK cells are asubset of null lymphocytes distinct from the NK and CTL populations.Activated macrophages can kill tumor cells in a manner that is notantigen dependent nor MHC restricted once activated. Activatedmacrophages are through to decrease the growth rate of the tumors theyinfiltrate. In vitro assays have identified other immune mechanisms suchas antibody-dependent, cell-mediated cytotoxic reactions and lysis byantibody plus complement. However, these immune effector mechanisms arethought to be less important in vivo than the function of NK, CTLs, LAK,and macrophages in vivo (for review see Piessens, W. F., and David, J.,“Tumor Immunology”, In: Scientific American Medicine, Vol. 2, ScientificAmerican Books, N.Y., pp. 1-13, 1996.

The goal of immunotherapy is to augment a patient's immune response toan established tumor. One method of immunotherapy includes the use ofadjuvants. Adjuvant substances derived from microorganisms, such asbacillus Calmette-Guerin, heighten the immune response and enhanceresistance to tumors in animals.

An “antigen” as used herein is a molecule capable of provoking an immuneresponse. Antigens include but are not limited to cells, cell extracts,proteins, polypeptides, peptides, polysaccharides, polysaccharideconjugates, peptide and non-peptide mimics of polysaccharides and othermolecules, small molecules, lipids, glycolipids, carbohydrates, virusesand viral extracts and multicellular organisms such as parasites andallergens. The term antigen broadly includes any type of molecule whichis recognized by a host immune system as being foreign. Antigens includebut are not limited to cancer antigens, microbial antigens, andallergens.

A “microbial antigen” as used herein is an antigen of a microorganismand includes but is not limited to virus, bacteria, parasites, andfungi. Such antigens include the intact microorganism as well as naturalisolates and fragments or derivatives thereof and also syntheticcompounds which are identical to or similar to natural microorganismantigens and induce an immune response specific for that microorganism.A compound is similar to a natural microorganism antigen if it inducesan immune response (humoral and/or cellular) to a natural microorganismantigen. Such antigens are used routinely in the art and are well knownto those of ordinary skill in the art.

A “cancer antigen” as used herein is a compound, such as a peptide orprotein, present in a tumor or cancer cell and which is capable ofprovoking an immune response when expressed on the surface of an antigenpresenting cell in the context of an MHC molecule. Cancer antigens canbe prepared from cancer cells either by preparing crude extracts ofcancer cells, for example, as described in Cohen, et al., 1994, CancerResearch, 54:1055, by partially purifying the antigens, by recombinanttechnology, or by de novo synthesis of known antigens. Cancer antigensinclude but are not limited to antigens that are recombinantlyexpressed, an immunogenic portion of, or a whole tumor or cancer. Suchantigens can be isolated or prepared recombinantly or by any other meansknown in the art.

Cancer or tumor antigens are differentially expressed by cancer cellsand can thereby be exploited in order to target cancer cells. Some ofthese antigens are encoded, although not necessarily expressed, bynormal cells. These antigens can be characterized as those which arenormally silent (i.e., not expressed) in normal cells, those that areexpressed only at certain stages of differentiation and those that aretemporally expressed such as embryonic and fetal antigens. Other cancerantigens are encoded by mutant cellular genes, such as oncogenes (e.g.,activated ras oncogene), suppressor genes (e.g., mutant p53), fusionproteins resulting from internal deletions or chromosomaltranslocations. Still other cancer antigens can be encoded by viralgenes such as those carried on RNA and DNA tumor viruses.

In some aspects of the invention, the subject is “exposed to” theantigen. As used herein, the term “exposed to” refers to either theactive step of contacting the subject with an antigen or the passiveexposure of the subject to the antigen in vivo. Methods for the activeexposure of a subject to an antigen are well-known in the art. Ingeneral, an antigen is administered directly to the subject by any meanssuch as intravenous, intramuscular, oral, transdermal, mucosal,intranasal, intratracheal, or subcutaneous administration. The antigencan be administered systemically or locally. Methods for administeringthe antigen and the immunostimulatory nucleic acid are described in moredetail below. A subject is passively exposed to an antigen if an antigenbecomes available for exposure to the immune cells in the body. Asubject may be passively exposed to an antigen, for instance, by entryof a foreign pathogen into the body or by the development of a tumorcell expressing a foreign antigen on its surface.

Active exposure of the antigen can occur at any time relative to theadministration of the immunostimulatory nucleic acid, including priorto, simultaneous with, or following nucleic acid administration. In someembodiments, the nucleic acid is administered at the same time, orsubstantially the same time (e.g., within an hour) of exposure to theantigen, but is administered in a different formulation. As an example,the antigen may be administered locally, and the nucleic acid may beadministered systemically, or vice versa.

The methods in which a subject is passively exposed to an antigen can beparticularly dependent on timing of administration of theimmunostimulatory nucleic acid. For instance, in a subject at risk ofdeveloping a cancer or an infectious disease or an allergic or asthmaticresponse, the subject may be administered the immunostimulatory nucleicacid on a regular basis when that risk is greatest, i.e., during allergyseason or after exposure to a cancer causing agent. Additionally theimmunostimulatory nucleic acid may be administered to travelers beforethey travel to foreign lands where they are at risk of exposure toinfectious agents. Likewise the immunostimulatory nucleic acid may beadministered to soldiers or civilians at risk of exposure to biowarfareto induce a systemic or mucosal immune response to the antigen when andif the subject is exposed to it.

A subject preferably is a non-rodent subject. A non-rodent subject shallmean a human or vertebrate animal including but not limited to a dog,cat, horse, cow, pig, sheep, goat, chicken, primate, e.g., monkey, andfish (aquaculture species), e.g. salmon, but specifically excludingrodents such as rats and mice.

Antigens can be derived from various sources including tumor, non-tumorcancers, allergens, and infectious pathogens. Each of the lists recitedherein is not intended to be limiting.

Examples of viruses that have been found in humans include but are notlimited to: Retroviridae (e.g. human immunodeficiency viruses, such asHIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III;and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses,hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses,echoviruses); Calciviridae (e.g. strains that cause gastroenteritis);Togaviridae (e.g. equine encephalitis viruses, rubella viruses);Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow feverviruses); Coronoviridae (e.g. coronaviruses); Rhabdoviradae (e.g.vesicular stomatitis viruses, rabies viruses); Coronaviridae (e.g.coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabiesviruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g.parainfluenza viruses, mumps virus, measles virus, respiratory syncytialvirus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g.Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses,orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis Bvirus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses,polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae(herpes simplex virus (HSV) 1 and 2, varicella zoster virus,cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swinefever virus); and unclassified viruses (e.g. the etiological agents ofSpongiform encephalopathies, the agent of delta hepatitis (thought to bea defective satellite of hepatitis B virus), the agents of non-A, non-Bhepatitis (class 1=internally transmitted; class 2=parenterallytransmitted (i.e. Hepatitis C); Norwalk and related viruses, andastroviruses).

Although many of the microbial antigens described herein relate to humandisorders, the invention is also useful for treating other non-humanvertebrates. Non-human vertebrates are also capable of developinginfections which can be prevented or treated with the immunostimulatorynucleic acids disclosed herein. For instance, in addition to thetreatment of infectious human diseases, the methods of the invention areuseful for treating infections of animals.

Both gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to, Pasteurella species, Staphylococci species, andStreptococcus species. Gram negative bacteria include, but are notlimited to, Escherichia coli, Pseudomonas species, and Salmonellaspecies. Specific examples of infectious bacteria include but are notlimited to, Helicobacter pyloris, Borelia burgdorferi, Legionellapneumophilia, Mycobacteria sps (e.g. M. tuberculosis, M. avium, M.intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus,Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes,Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae(Group B Streptococcus), Streptococcus (viridans group), Streptococcusfaecalis, Streptococcus bovis, Streptococcus (anaerobic sps.),Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcussp., Haemophilus influenzae, Bacillus antracis, corynebacteriumdiphtheriae, corynebacterium sp., Erysipelothrix rhusiopathiae,Clostridium perfringers, Clostridium tetani, Enterobacter aerogenes,Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp.,Fusobacterium nucleatum, Streptobacillus moniliformis, Treponemapallidium, Treponema pertenue, Leptospira, Rickettsia, and Actinomycesisraelli.

Polypeptides of bacterial pathogens include but are not limited to aniron-regulated outer membrane protein, (IROMP), an outer membraneprotein (OMP), and an A-protein of Aeromonis salmonicida which causesfurunculosis, p57 protein of Renibacterium salmoninarum which causesbacterial kidney disease (BKD), major surface associated antigen (msa),a surface expressed cytotoxin (mpr), a surface expressed hemolysin(ish), and a flagellar antigen of Yersiniosis; an extracellular protein(ECP), an iron-regulated outer membrane protein (IROMP), and astructural protein of Pasteurellosis; an OMP and a flagellar protein ofVibrosis anguillarum and V. ordalii; a flagellar protein, an OMPprotein, aroA, and purA of Edwardsiellosis ictaluri and E. tarda; andsurface antigen of Ichthyophthirius; and a structural and regulatoryprotein of Cytophaga columnari; and a structural and regulatory proteinof Rickettsia.

Polypeptides of a parasitic pathogen include but are not limited to thesurface antigens of Ichthyophthirius.

Examples of fungi include Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans. Other infectious organisms (i.e.,protists) include Plasmodium spp. such as Plasmodium falciparum,Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax andToxoplasma gondii. Blood-borne and/or tissues parasites includePlasmodium spp., Babesia microti, Babesia divergens, Leishmania tropica,Leishmania spp., Leishmania braziliensis, Leishmania donovani,Trypanosoma gambiense and Trypanosoma rhodesiense (African sleepingsickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasma gondii.

Other medically relevant microorganisms have been described extensivelyin the literature, e.g., see C. G. A Thomas, Medical Microbiology,Bailliere Tindall, Great Britain 1983, the entire contents of which ishereby incorporated by reference.

Many vaccines for the treatment of non-human vertebrates are disclosedin Bennett, K. Compendium of Veterinary Products, 3rd ed. North AmericanCompendiums, Inc., 1995. As discussed above, antigens include infectiousmicrobes such as virus, parasite, bacteria and fungi and fragmentsthereof, derived from natural sources or synthetically. Infectiousviruses of both human and non-human vertebrates, include retroviruses,RNA viruses and DNA viruses. This group of retroviruses includes bothsimple retroviruses and complex retroviruses. The simple retrovirusesinclude the subgroups of B-type retroviruses, C-type retroviruses andD-type retroviruses. An example of a B-type retrovirus is mouse mammarytumor virus (MMTV). The C-type retroviruses include subgroups C-typegroup A (including Rous sarcoma virus (RSV), avian leukemia virus (ALV),and avian myeloblastosis virus (AMV)) and C-type group B (includingfeline leukemia virus (FeLV), gibbon ape leukemia virus (GALV), spleennecrosis virus (SNV), reticuloendotheliosis virus (RV) and simiansarcoma virus (SSV)). The D-type retroviruses include Mason-Pfizermonkey virus (MPMV) and simian retrovirus type 1 (SRV-1). The complexretroviruses include the subgroups of lentiviruses, T-cell leukemiaviruses and the foamy viruses. Lentiviruses include HIV-1, but alsoinclude HIV-2, SIV, Visna virus, feline immunodeficiency virus (FIV),and equine infectious anemia virus (EIAV). The T-cell leukemia virusesinclude HTLV-1, HTLV-II, simian T-cell leukemia virus (STLV), and bovineleukemia virus (BLV). The foamy viruses include human foamy virus (HFV),simian foamy virus (SFV) and bovine foamy virus (BFV).

Examples of other RNA viruses that are antigens in vertebrate animalsinclude, but are not limited to, members of the family Reoviridae,including the genus Orthoreovirus (multiple serotypes of both mammalianand avian retroviruses), the genus Orbivirus (Bluetongue virus,Eugenangee virus, Kemerovo virus, African horse sickness virus, andColorado Tick Fever virus), the genus Rotavirus (human rotavirus,Nebraska calf diarrhea virus, simian rotavirus, bovine or ovinerotavirus, avian rotavirus); the family Picornaviridae, including thegenus Enterovirus (poliovirus, Coxsackie virus A and B, entericcytopathic human orphan (ECHO) viruses, hepatitis A virus, Simianenteroviruses, Murine encephalomyelitis (ME) viruses, Poliovirus muris,Bovine enteroviruses, Porcine enteroviruses, the genus Cardiovirus(Encephalomyocarditis virus (EMC), Mengovirus), the genus Rhinovirus(Human rhinoviruses including at least 113 subtypes; otherrhinoviruses), the genus Apthovirus (Foot and Mouth disease (FMDV); thefamily Calciviridae, including Vesicular exanthema of swine virus, SanMiguel sea lion virus, Feline picornavirus and Norwalk virus; the familyTogaviridae, including the genus Alphavirus (Eastern equine encephalitisvirus, Semliki forest virus, Sindbis virus, Chikungunya virus,O'Nyong-Nyong virus, Ross river virus, Venezuelan equine encephalitisvirus, Western equine encephalitis virus), the genus Flavirius (Mosquitoborne yellow fever virus, Dengue virus, Japanese encephalitis virus, St.Louis encephalitis virus, Murray Valley encephalitis virus, West Nilevirus, Kunjin virus, Central European tick borne virus, Far Eastern tickborne virus, Kyasanur forest virus, Louping III virus, Powassan virus,Omsk hemorrhagic fever virus), the genus Rubivirus (Rubella virus), thegenus Pestivirus (Mucosal disease virus, Hog cholera virus, Borderdisease virus); the family Bunyaviridae, including the genus Bunyvirus(Bunyamwera and related viruses, California encephalitis group viruses),the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fevervirus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus,Nairobi sheep disease virus), and the genus Uukuvirus (Uukuniemi andrelated viruses); the family Orthomyxoviridae, including the genusInfluenza virus (Influenza virus type A, many human subtypes); Swineinfluenza virus, and Avian and Equine Influenza viruses; influenza typeB (many human subtypes), and influenza type C (possible separate genus);the family paramyxoviridae, including the genus Paramyxovirus(Parainfluenza virus type 1, Sendai virus, Hemadsorption virus,Parainfluenza viruses types 2 to 5, Newcastle Disease Virus, Mumpsvirus), the genus Morbillivirus (Measles virus, subacute sclerosingpanencephalitis virus, distemper virus, Rinderpest virus), the genusPneumovirus (respiratory syncytial virus (RSV), Bovine respiratorysyncytial virus and Pneumonia virus); the family Rhabdoviridae,including the genus Vesiculovirus (VSV), Chandipura virus, Flanders-HartPark virus), the genus Lyssavirus (Rabies virus), fish Rhabdoviruses,and two probable Rhabdoviruses (Marburg virus and Ebola virus); thefamily Arenaviridae, including Lymphocytic choriomeningitis virus (LCM),Tacaribe virus complex, and Lassa virus; the family Coronoaviridae,including Infectious Bronchitis Virus (IBV), Hepatitis virus, Humanenteric corona virus, and Feline infectious peritonitis (Felinecoronavirus).

Illustrative DNA viruses that are antigens in vertebrate animalsinclude, but are not limited to, the family Poxyiridae, including thegenus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia,Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus(Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avianpoxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genusSuipoxvirus (Swinepox), the genus Parapoxvirus (contagious postulardermatitis virus, pseudocowpox, bovine papular stomatitis virus); thefamily Iridoviridae (African swine fever virus, Frog viruses 2 and 3,Lymphocystis virus of fish); the family Herpesviridae, including thealpha-Herpesviruses (Herpes Simplex Types 1 and 2, Varicella-Zoster,Equine abortion virus, Equine herpes virus 2 and 3, pseudorabies virus,infectious bovine keratoconjunctivitis virus, infectious bovinerhinotracheitis virus, feline rhinotracheitis virus, infectiouslaryngotracheitis virus) the Beta-herpesviruses (Human cytomegalovirusand cytomegaloviruses of swine and monkeys); the gamma-herpesviruses(Epstein-Barr virus (EBV), Marek's disease virus, Herpes saimiri,Herpesvirus ateles, Herpesvirus sylvilagus, guinea pig herpes virus,Lucke tumor virus); the family Adenoviridae, including the genusMastadenovirus (Human subgroups A,B,C,D,E and ungrouped; simianadenoviruses (at least 23 serotypes), infectious canine hepatitis, andadenoviruses of cattle, pigs, sheep, frogs and many other species, thegenus Aviadenovirus (Avian adenoviruses); and non-cultivatableadenoviruses; the family Papoviridae, including the genus Papillomavirus(Human papilloma viruses, bovine papilloma viruses, Shope rabbitpapilloma virus, and various pathogenic papilloma viruses of otherspecies), the genus Polyomavirus (polyomavirus, Simian vacuolating agent(SV-40), Rabbit vacuolating agent (RKV), K virus, BK virus, JC virus,and other primate polyoma viruses such as Lymphotrophic papillomavirus); the family Parvoviridae including the genus Adeno-associatedviruses, the genus Parvovirus (Feline panleukopenia virus, bovineparvovirus, canine parvovirus, Aleutian mink disease virus, etc).Finally, DNA viruses may include viruses which do not fit into the abovefamilies such as Kuru and Creutzfeldt-Jacob disease viruses and chronicinfectious neuropathic agents (CHINA virus).

The immunostimulatory nucleic acids can also be used to induce an immuneresponse, such as an antigen specific immune response, birds such ashens, chickens, turkeys, ducks, geese, quail, and pheasant. Birds areprime targets for many types of infections.

Hatching birds are exposed to pathogenic microorganisms shortly afterbirth. Although these birds are initially protected against pathogens bymaternal derived antibodies, this protection is only temporary, and thebird's own immature immune system must begin to protect the bird againstthe pathogens. It is often desirable to prevent infection in young birdswhen they are most susceptible. It is also desirable to prevent againstinfection in older birds, especially when the birds are housed in closedquarters, leading to the rapid spread of disease. Thus, it is desirableto administer the Immunostimulatory nucleic acid and the non-nucleicacid adjuvant of the invention to birds to enhance an antigen-specificimmune response when antigen is present.

An example of a common infection in chickens is chicken infectiousanemia virus (CIAV). CIAV was first isolated in Japan in 1979 during aninvestigation of a Marek's disease vaccination break (Yuasa et al.,1979, Avian Dis. 23:366-385). Since that time, CIAV has been detected incommercial poultry in all major poultry producing countries (van Bulowet al., 1991, pp.690-699) in Diseases of Poultry, 9th edition, IowaState University Press).

CIAV infection results in a clinical disease, characterized by anemia,hemorrhage and immunosuppression, in young susceptible chickens. Atrophyof the thymus and of the bone marrow and consistent lesions ofCIAV-infected chickens are also characteristic of CIAV infection.Lymphocyte depletion in the thymus, and occasionally in the bursa ofFabricius, results in immunosuppression and increased susceptibility tosecondary viral, bacterial, or fungal infections which then complicatethe course of the disease. The immunosuppression may cause aggravateddisease after infection with one or more of Marek's disease virus (MDV),infectious bursal disease virus, reticuloendotheliosis virus,adenovirus, or reovirus. It has been reported that pathogenesis of MDVis enhanced by CIAV (DeBoer et al., 1989, p. 28 In Proceedings of the38th Western Poultry Diseases Conference, Tempe, Ariz.). Further, it hasbeen reported that CIAV aggravates the signs of infectious bursaldisease (Rosenberger et al., 1989, Avian Dis. 33:707-713). Chickensdevelop an age resistance to experimentally induced disease due to CAA.This is essentially complete by the age of 2 weeks, but older birds arestill susceptible to infection (Yuasa, N. et al., 1979 supra; Yuasa, N.et al., Arian Diseases 24, 202-209, 1980). However, if chickens aredually infected with CAA and an immunosuppressive agent (IBDV, MDVetc.), age resistance against the disease is delayed (Yuasa, N. et al.,1979 and 1980 supra; Bulow von V. et al., J. Veterinary Medicine 33,93-116, 1986). Characteristics of CIAV that may potentiate diseasetransmission include high resistance to environmental inactivation andsome common disinfectants. The economic impact of CIAV infection on thepoultry industry is clear from the fact that 10% to 30% of infectedbirds in disease outbreaks die.

Vaccination of birds, like other vertebrate animals can be performed atany age. Normally, vaccinations are performed at up to 12 weeks of agefor a live microorganism and between 14-18 weeks for an inactivatedmicroorganism or other type of vaccine. For in ovo vaccination,vaccination can be performed in the last quarter of embryo development.The vaccine may be administered subcutaneously, by spray, orally,intraocularly, intratracheally, nasally, or by other mucosal deliverymethods described herein. Thus, the immunostimulatory nucleic acids ofthe invention can be administered to birds and other non-humanvertebrates using routine vaccination schedules and the antigen can beadministered after an appropriate time period as described herein.

Cattle and livestock are also susceptible to infection. Diseases whichaffect these animals can produce severe economic losses, especiallyamongst cattle. The methods of the invention can be used to protectagainst infection in livestock, such as cows, horses, pigs, sheep, andgoats.

Cows can be infected by bovine viruses. Bovine viral diarrhea virus(BVDV) is a small enveloped positive-stranded RNA virus and isclassified, along with hog cholera virus (HOCV) and sheep border diseasevirus (BDV), in the pestivirus genus. Although, Pestiviruses werepreviously classified in the Togaviridae family, some studies havesuggested their reclassification within the Flaviviridae family alongwith the flavivirus and hepatitis C virus (HCV) groups (Francki, et al.,1991).

BVDV, which is an important pathogen of cattle can be distinguished,based on cell culture analysis, into cytopathogenic (CP) andnoncytopathogenic (NCP) biotypes. The NCP biotype is more widespreadalthough both biotypes can be found in cattle. If a pregnant cow becomesinfected with an NCP strain, the cow can give birth to a persistentlyinfected and specifically immunotolerant calf that will spread virusduring its lifetime. The persistently infected cattle can succumb tomucosal disease and both biotypes can then be isolated from the animal.Clinical manifestations can include abortion, teratogenesis, andrespiratory problems, mucosal disease and mild diarrhea. In addition,severe thrombocytopenia, associated with herd epidemics, that may resultin the death of the animal has been described and strains associatedwith this disease seem more virulent than the classical BVDVs.

Equine herpes viruses (EHV) comprise a group of antigenically distinctbiological agents which cause a variety of infections in horses rangingfrom subclinical to fatal disease. These include Equine herpesvirus-1(EHV-1), a ubiquitous pathogen in horses. EHV-1 is associated withepidemics of abortion, respiratory tract disease, and central nervoussystem disorders. Primary infection of upper respiratory tract of younghorses results in a febrile illness which lasts for 8 to 10 days.Immunologically experienced mares may be re-infected via the respiratorytract without disease becoming apparent, so that abortion usually occurswithout warning. The neurological syndrome is associated withrespiratory disease or abortion and can affect animals of either sex atany age, leading to lack of co-ordination, weakness and posteriorparalysis (Telford, E. A. R. et al., Virology 189, 304-316, 1992). OtherEHV's include EHV-2, or equine cytomegalovirus, EHV-3, equine coitalexanthema virus, and EHV-4, previously classified as EHV-1 subtype 2.

Sheep and goats can be infected by a variety of dangerous microorganismsincluding visna-maedi.

Primates such as monkeys, apes and macaques can be infected by simianimmunodeficiency virus. Inactivated cell-virus and cell-free wholesimian immunodeficiency vaccines have been reported to afford protectionin macaques (Stott et al. (1990) Lancet 36:1538-1541; Desrosiers et al.PNAS USA (1989) 86:6353-6357; Murphey-Corb et al. (1989) Science246:1293-1297; and Carlson et al. (1990) AIDS Res. Human Retroviruses6:1239-1246). A recombinant HIV gp120 vaccine has been reported toafford protection in chimpanzees (Berman et al. (1990) Nature345:622-625).

Cats, both domestic and wild, are susceptible to infection with avariety of microorganisms. For instance, feline infectious peritonitisis a disease which occurs in both domestic and wild cats, such as lions,leopards, cheetahs, and jaguars. When it is desirable to preventinfection with this and other types of pathogenic organisms in cats, themethods of the invention can be used to vaccinate cats to protect themagainst infection.

Domestic cats may become infected with several retroviruses, includingbut not limited to feline leukemia virus (FeLV), feline sarcoma virus(FeSV), endogenous type Concornavirus (RD-114), and felinesyncytia-forming virus (FeSFV). Of these, FeLV is the most significantpathogen, causing diverse symptoms, including lymphoreticular andmyeloid neoplasms, anemias, immune mediated disorders, and animmunodeficiency syndrome which is similar to human acquired immunedeficiency syndrome (AIDS). Recently, a particular replication-defectiveFeLV mutant, designated FeLV-AIDS, has been more particularly associatedwith immunosuppressive properties.

The discovery of feline T-lymphotropic lentivirus (also referred to asfeline immunodeficiency) was first reported in Pedersen et al. (1987)Science 235:790-793. Characteristics of FIV have been reported inYamamoto et al. (1988) Leukemia, December Supplement 2:204S-215S;Yamamoto et al. (1988) Am. J. Vet. Res. 49:1246-1258; and Ackley et al.(1990) J. Virol. 64:5652-5655. Cloning and sequence analysis of FIV havebeen reported in Olmsted et al. (1989) Proc. Natl. Acad. Sci. USA86:2448-2452 and 86:4355-4360.

Feline infectious peritonitis (FIP) is a sporadic disease occurringunpredictably in domestic and wild Felidae. While FIP is primarily adisease of domestic cats, it has been diagnosed in lions, mountainlions, leopards, cheetahs, and the jaguar. Smaller wild cats that havebeen afflicted with FIP include the lynx and caracal, sand cat, andpallas cat. In domestic cats, the disease occurs predominantly in younganimals, although cats of all ages are susceptible. A peak incidenceoccurs between 6 and 12 months of age. A decline in incidence is notedfrom 5 to 13 years of age, followed by an increased incidence in cats 14to 15 years old.

Viral, bacterial, and parasitic diseases in fin-fish, shellfish or otheraquatic life forms pose a serious problem for the aquaculture industry.Owing to the high density of animals in the hatchery tanks or enclosedmarine farming areas, infectious diseases may eradicate a largeproportion of the stock in, for example, a fin-fish, shellfish, or otheraquatic life forms facility. Prevention of disease is a more desiredremedy to these threats to fish than intervention once the disease is inprogress. Vaccination of fish is the only preventative method which mayoffer long-term protection through immunity. Nucleic acid basedvaccinations are described in U.S. Pat. No. 5,780,448 issued to Davis.

The fish immune system has many features similar to the mammalian immunesystem, such as the presence of B cells, T cells, lymphokines,complement, and immunoglobulins. Fish have lymphocyte subclasses withroles that appear similar in many respects to those of the B and T cellsof mammals. Vaccines can be administered by immersion or orally.

Aquaculture species include but are not limited to fin-fish, shellfish,and other aquatic animals. Fin-fish include all vertebrate fish, whichmay be bony or cartilaginous fish, such as, for example, salmonids,carp, catfish, yellowtail, seabream, and seabass. Salmonids are a familyof fin-fish which include trout (including rainbow trout), salmon, andArctic char. Examples of shellfish include, but are not limited to,clams, lobster, shrimp, crab, and oysters. Other cultured aquaticanimals include, but are not limited to eels, squid, and octopi.

Polypeptides of viral aquaculture pathogens include but are not limitedto glycoprotein (G) or nucleoprotein (N) of viral hemorrhagic septicemiavirus (VHSV); G or N proteins of infectious hematopoietic necrosis virus(IHNV); VP1, VP2, VP3 or N structural proteins of infectious pancreaticnecrosis virus (IPNV); G protein of spring viremia of carp (SVC); and amembrane-associated protein, tegumin or capsid protein or glycoproteinof channel catfish virus (CCV).

Typical parasites infecting horses are Gasterophilus spp.; Eimerialeuckarti, Giardia spp.; Tritrichomonas equi; Babesia spp. (RBC's),Theileria equi; Trypanosoma spp.; Klossiella equi; Sarcocystis spp.

Typical parasites infecting swine include Eimeria bebliecki, Eimeriascabra, Isospora suis, Giardia spp.; Balantidium coli, Entamoebahistolytica; Toxoplasma gondii and Sarcocystis spp., and Trichinellaspiralis.

The major parasites of dairy and beef cattle include Eimeria spp.,Cryptosporidium sp., Giardia spp.; Toxoplasma gondii; Babesia bovis(RBC), Babesia bigemina (RBC), Trypanosoma spp. (plasma), Theileria spp.(RBC); Theileria parva (lymphocytes); Tritrichomonas foetus; andSarcocystis spp.

The major parasites of raptors include Trichomonas gallinae; Coccidia(Eimeria spp.); Plasmodium relictum, Leucocytozoon danilewskyi (owls),Haemoproteus spp., Trypanosoma spp.; Histomonas; Cryptosporidiummeleagridis, Cryptosporidium baileyi, Giardia, Eimeria; Toxoplasma.

Typical parasites infecting sheep and goats include Eimeria spp.,Cryptosporidium sp., Giardia sp.; Toxoplasma gondii; Babesia spp. (RBC),Trypanosoma spp. (plasma), Theileria spp. (RBC); and Sarcocystis spp.

Typical parasitic infections in poultry include coccidiosis caused byEimeria acervulina, E. necatrix, E. tenella, Isospora spp. and Eimeriatruncata; histomoniasis, caused by Histomonas meleagridis and Histomonasgallinarum; trichomoniasis caused by Trichomonas gallinae; andhexamitiasis caused by Hexamita meleagridis. Poultry can also beinfected Emeria maxima, Emeria meleagridis, Eimeria adenoeides, Eimeriameleagrimitis, Cryptosporidium, Eimeria brunetti, Emeria adenoeides,Leucocytozoon spp., Plasmodium spp., Hemoproteus meleagridis, Toxoplasmagondii and Sarcocystis.

The methods of the invention can also be applied to the treatment and/orprevention of parasitic infection in dogs, cats, birds, fish andferrets. Typical parasites of birds include Trichomonas gallinae;Eimeria spp., Isospora spp., Giardia; Cryptosporidium; Sarcocystis spp.,Toxoplasma gondii, Haemoproteus/Parahaemoproteus, Plasmodium spp.,Leucocytozoon/Akiba, Atoxoplasma, Trypanosoma spp. Typical parasitesinfecting dogs include Trichinella spiralis; Isopora spp., Sarcocystisspp., Cryptosporidium spp., Hammondia spp., Giardia duodenalis (canis);Balantidium coli, Entamoeba histolytica; Hepatozoon canis; Toxoplasmagondii, Trypanosoma cruzi; Babesia canis; Leishmania amastigotes;Neospora caninum.

Typical parasites infecting feline species include Isospora spp.,Toxoplasma gondii, Sarcocystis spp., Hammondia hammondi, Besnoitia spp.,Giardia spp.; Entamoeba histolytica; Hepatozoon canis, Cytauxzoon sp.,Cytauxzoon sp., Cytauxzoon sp. (red cells, RE cells).

Typical parasites infecting fish include Hexamita spp., Eimeria spp.;Cryptobia spp., Nosema spp., Myxosoma spp., Chilodonella spp.,Trichodina spp.; Plistophora spp., Myxosoma Henneguya; Costia spp.,Ichthyophithirius spp., and Oodinium spp.

Typical parasites of wild mammals include Giardia spp. (carnivores,herbivores), Isospora spp. (carnivores), Eimeria spp. (carnivores,herbivores); Theileria spp. (herbivores), Babesia spp. (carnivores,herbivores), Trypanosoma spp. (carnivores, herbivores); Schistosoma spp.(herbivores); Fasciola hepatica (herbivores), Fascioloides magna(herbivores), Fasciola gigantica (herbivores), Trichinella spiralis(carnivores, herbivores).

Parasitic infections in zoos can also pose serious problems. Typicalparasites of the bovidae family (blesbok, antelope, banteng, eland,gaur, impala, klipspringer, kudu, gazelle) include Eimeria spp. Typicalparasites in the pinnipedae family (seal, sea lion) include Eimeriaphocae. Typical parasites in the camelidae family (camels, llamas)include Eimeria spp. Typical parasites of the giraffidae family(giraffes) include Eimeria spp. Typical parasites in the elephantidaefamily (African and Asian) include Fasciola spp. Typical parasites oflower primates (chimpanzees, orangutans, apes, baboons, macaques,monkeys) include Giardia sp.; Balantidium coli, Entamoeba histolytica,Sarcocystis spp., Toxoplasma gondii; Plasmodim spp. (RBC), Babesia spp.(RBC), Trypanosoma spp. (plasma), Leishmania spp. (macrophages).

Cancer is one of the leading causes of death in companion animals (i.e.,cats and dogs). Cancer usually strikes older animals which, in the caseof house pets, have become integrated into the family. Forty-five % ofdogs older than 10 years of age, are likely to succumb to the disease.The most common treatment options include surgery, chemotherapy andradiation therapy. Others treatment modalities which have been used withsome success are laser therapy, cryotherapy, hyperthermia andimmunotherapy. The choice of treatment depends on type of cancer anddegree of dissemination. Unless the malignant growth is confined to adiscrete area in the body, it is difficult to remove only malignanttissue without also affecting normal cells.

Malignant disorders commonly diagnosed in dogs and cats include but arenot limited to lymphosarcoma, osteosarcoma, mammary tumors, mastocytoma,brain tumor, melanoma, adenosquamous carcinoma, carcinoid lung tumor,bronchial gland tumor, bronchiolar adenocarcinoma, fibroma,myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma,retinoblastoma, Ewing's sarcoma, Wilm's tumor, Burkitt's lymphoma,microglioma, neuroblastoma, osteoclastoma, oral neoplasia, fibrosarcoma,osteosarcoma and rhabdomyosarcoma. Other neoplasias in dogs includegenital squamous cell carcinoma, transmissable venereal tumor,testicular tumor, seminoma, Sertoli cell tumor, hemangiopericytoma,histiocytoma, chloroma (granulocytic sarcoma), corneal papilloma,corneal squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma,basal cell tumor, thymoma, stomach tumor, adrenal gland carcinoma, oralpapillomatosis, hemangioendothelioma and cystadenoma. Additionalmalignancies diagnosed in cats include follicular lymphoma, intestinallymphosarcoma, fibrosarcoma and pulmonary squamous cell carcinoma. Theferret, an ever-more popular house pet is known to develop insulinoma,lymphoma, sarcoma, neuroma, pancreatic islet cell tumor, gastric MALTlymphoma and gastric adenocarcinoma.

Neoplasias affecting agricultural livestock include leukemia,hemangiopericytoma and bovine ocular neoplasia (in cattle); preputialfibrosarcoma, ulcerative squamous cell carcinoma, preputial carcinoma,connective tissue neoplasia and mastocytoma (in horses); hepatocellularcarcinoma (in swine); lymphoma and pulmonary adenomatosis (in sheep);pulmonary sarcoma, lymphoma, Rous sarcoma, reticulendotheliosis,fibrosarcoma, nephroblastoma, B-cell lymphoma and lymphoid leukosis (inavian species); retinoblastoma, hepatic neoplasia, lymphosarcoma(lymphoblastic lymphoma), plasmacytoid leukemia and swimbladder sarcoma(in fish), caseous lumphadenitis (CLA): chronic, infectious, contagiousdisease of sheep and goats caused by the bacterium Corynebacteriumpseudotuberculosis, and contagious lung tumor of sheep caused byjaagsiekte.

An allergen refers to a substance (antigen) that can induce an allergicor asthmatic response in a susceptible subject. The list of allergens isenormous and can include pollens, insect venoms, animal dander dust,fungal spores and drugs (e.g. penicillin). Examples of natural, animaland plant allergens include but are not limited to proteins specific tothe following genuses: Canine (Canis familiaris); Dermatophagoides (e.g.Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosiaartemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum);Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata);Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus(Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris);Plantago (e.g. Plantago lanceolata); Parietaria (e.g. Parietariaofficinalis or Parietaria judaica); Blattella (e.g. Blattellagermanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressussempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus(e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis andJuniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g.Chamaecyparis obtusa); Periplaneta (e.g. Periplanetaamericana);Agropyron (e.g. Agropyron repens); Secale (e.g. Secalecereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylisglomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis orPoa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus);Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g.Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum (e.g.Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum(e.g.Paspalum notatum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g.Bromus inermis).

The antigen may be an antigen that is encoded by a nucleic acid vectoror it may be not encoded in a nucleic acid vector. In the former casethe nucleic acid vector is administered to the subject and the antigenis expressed in vivo. In the latter case the antigen may be administereddirectly to the subject. An antigen not encoded in a nucleic acid vectoras used herein refers to any type of antigen that is not a nucleic acid.For instance, in some aspects of the invention the antigen not encodedin a nucleic acid vector is a polypeptide. Minor modifications of theprimary amino acid sequences of polypeptide antigens may also result ina polypeptide which has substantially equivalent antigenic activity ascompared to the unmodified counterpart polypeptide. Such modificationsmay be deliberate, as by site-directed mutagenesis, or may bespontaneous. All of the polypeptides produced by these modifications areincluded herein as long as antigenicity still exists. The polypeptidemay be, for example, a viral polypeptide.

The term “substantially purified” as used herein refers to a polypeptidewhich is substantially free of other proteins, lipids, carbohydrates orother materials with which it is naturally associated. One skilled inthe art can purify viral or bacterial polypeptides using standardtechniques for protein purification. The substantially pure polypeptidewill often yield a single major band on a non-reducing polyacrylamidegel. In the case of partially glycosylated polypeptides or those thathave several start codons, there may be several bands on a non-reducingpolyacrylamide gel, but these will form a distinctive pattern for thatpolypeptide. The purity of the viral or bacterial polypeptide can alsobe determined by amino-terminal amino acid sequence analysis. Othertypes of antigens not encoded by a nucleic acid vector such aspolysaccharides, small molecule, mimics etc are described above, andincluded within the invention.

The invention also utilizes polynucleotides encoding the antigenicpolypeptides. It is envisioned that the antigen may be delivered to thesubject in a nucleic acid molecule which encodes for the antigen suchthat the antigen must be expressed in vivo. Such antigens delivered tothe subject in a nucleic acid vector are referred to as antigens encodedby a nucleic acid vector. The nucleic acid encoding the antigen isoperatively linked to a gene expression sequence which directs theexpression of the antigen nucleic acid within a eukaryotic cell. Thegene expression sequence is any regulatory nucleotide sequence, such asa promoter sequence or promoter-enhancer combination, which facilitatesthe efficient transcription and translation of the antigen nucleic acidto which it is operatively linked. The gene expression sequence may, forexample, be a mammalian or viral promoter, such as a constitutive orinducible promoter. Constitutive mammalian promoters include, but arenot limited to, the promoters for the following genes: hypoxanthinephosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate kinase,b-actin promoter and other constitutive promoters. Exemplary viralpromoters which function constitutively in eukaryotic cells include, forexample, promoters from the cytomegalovirus (CMV), simian virus (e.g.,SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV),Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) ofMoloney leukemia virus and other retroviruses, and the thymidine kinasepromoter of herpes simplex virus. Other constitutive promoters are knownto those of ordinary skill in the art. The promoters useful as geneexpression sequences of the invention also include inducible promoters.Inducible promoters are expressed in the presence of an inducing agent.For example, the metallothionein promoter is induced to promotetranscription and translation in the presence of certain metal ions.Other inducible promoters are known to those of ordinary skill in theart.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined antigen nucleic acid. The gene expression sequences optionallyinclude enhancer sequences or upstream activator sequences as desired.

The antigen nucleic acid is operatively linked to the gene expressionsequence. As used herein, the antigen nucleic acid sequence and the geneexpression sequence are said to be operably linked when they arecovalently linked in such a way as to place the expression ortranscription and/or translation of the antigen coding sequence underthe influence or control of the gene expression sequence. Two DNAsequences are said to be operably linked if induction of a promoter inthe 5′ gene expression sequence results in the transcription of theantigen sequence and if the nature of the linkage between the two DNAsequences does not (1) result in the introduction of a frame-shiftmutation, (2) interfere with the ability of the promoter region todirect the transcription of the antigen sequence, or (3) interfere withthe ability of the corresponding RNA transcript to be translated into aprotein. Thus, a gene expression sequence would be operably linked to anantigen nucleic acid sequence if the gene expression sequence werecapable of effecting transcription of that antigen nucleic acid sequencesuch that the resulting transcript is translated into the desiredprotein or polypeptide.

The antigen nucleic acid of the invention may be delivered to the immunesystem alone or in association with a vector. In its broadest sense, avector is any vehicle capable of facilitating the transfer of theantigen nucleic acid to the cells of the immune system so that theantigen can be expressed and presented on the surface of the immunecell. The vector generally transports the nucleic acid to the immunecells with reduced degradation relative to the extent of degradationthat would result in the absence of the vector. The vector optionallyincludes the above-described gene expression sequence to enhanceexpression of the antigen nucleic acid in immune cells. In general, thevectors useful in the invention include, but are not limited to,plasmids, phagemids, viruses, other vehicles derived from viral orbacterial sources that have been manipulated by the insertion orincorporation of the antigen nucleic acid sequences. Viral vectors are apreferred type of vector and include, but are not limited to, nucleicacid sequences from the following viruses: retrovirus, such as Moloneymurine leukemia virus, Harvey murine sarcoma virus, murine mammary tumorvirus, and Rous sarcoma virus; adenovirus, adeno-associated virus;SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papillomaviruses; herpes virus; vaccinia virus; polio virus; and RNA virus suchas a retrovirus. One can readily employ other vectors not named butknown in the art.

Preferred viral vectors are based on non-cytopathic eukaryotic virusesin which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include retroviruses, the life cycle ofwhich involves reverse transcription of genomic viral RNA into DNA withsubsequent proviral integration into host cellular DNA. Retroviruseshave been approved for human gene therapy trials. Most useful are thoseretroviruses that are replication-deficient (i.e., capable of directingsynthesis of the desired proteins, but incapable of manufacturing aninfectious particle). Such genetically altered retroviral expressionvectors have general utility for the high-efficiency transduction ofgenes in vivo. Standard protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous geneticmaterial into a plasmid, transfection of a packaging cell lined withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with viral particles) are provided inKriegler, M., Gene Transfer and Expression, A Laboratory Manual W.H.Freeman C.O., New York (1990) and Murry, E. J. Methods in MolecularBiology, vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

A preferred virus for certain applications is the adeno-associatedvirus, a double-stranded DNA virus. The adeno-associated virus can beengineered to be replication-deficient and is capable of infecting awide range of cell types and species. It further has advantages such as,heat and lipid solvent stability; high transduction frequencies in cellsof diverse lineages, including hemopoietic cells; and lack ofsuperinfection inhibition thus allowing multiple series oftransductions. Reportedly, the adeno-associated virus can integrate intohuman cellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression characteristic of retroviral infection. In addition,wild-type adeno-associated virus infections have been followed in tissueculture for greater than 100 passages in the absence of selectivepressure, implying that the adeno-associated virus genomic integrationis a relatively stable event. The adeno-associated virus can alsofunction in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well-known to those of skill inthe art. See e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Inthe last few years, plasmid vectors have been found to be particularlyadvantageous for delivering genes to cells in vivo because of theirinability to replicate within and integrate into a host genome. Theseplasmids, however, having a promoter compatible with the host cell, canexpress a peptide from a gene operatively encoded within the plasmid.Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40,and pBlueScript. Other plasmids are well-known to those of ordinaryskill in the art. Additionally, plasmids may be custom designed usingrestriction enzymes and ligation reactions to remove and add specificfragments of DNA.

It has recently been discovered that gene carrying plasmids can bedelivered to the immune system using bacteria. Modified forms ofbacteria such as Salmonella can be transfected with the plasmid and usedas delivery vehicles. The bacterial delivery vehicles can beadministered to a host subject orally or by other administration means.The bacteria deliver the plasmid to immune cells, e.g. B cells,dendritic cells, likely by passing through the gut barrier. High levelsof immune protection have been established using this methodology. Suchmethods of delivery are useful for the aspects of the inventionutilizing systemic delivery of antigen, Immunostimulatory nucleic acidand/or other therapeutic agent.

Thus, in addition to being suitable as stand alone agents, theimmunostimulatory nucleic acids are useful, inter alia, as vaccineadjuvants. It was previously established that CpG oligonucleotides areexcellent vaccine adjuvants. In order to identify the bestimmunostimulatory nucleic acids for use as a vaccine adjuvant in humansand other non-rodent animals, in vivo screening of different nucleicacids for this purpose was conducted. Several in vitro assays wereevaluated in mice for their predictive value of adjuvant activity invivo. During the course of this study, an in vitro test that ispredictive of in vivo efficacy was identified. It was discovered, rathersurprisingly, that both B cell and NK cell activation correlatedparticularly well with the ability of an immunostimulatory nucleic acidto enhance an in vivo immune response against an antigen.

The nucleic acids are also useful for improving survival,differentiation, activation and maturation of dendritic cells. Theimmunostimulatory nucleic acids have the unique capability to promotecell survival, differentiation, activation and maturation of dendriticcells. Dendritic precursor cells isolated from blood by immunomagneticcell sorting develop morphologic and functional characteristics ofdendritic cells during a two day incubation with GM-CSF. Without GM-CSFthese cells undergo apoptosis. The immunostimulatory nucleic acids aresuperior to GM-CSF in promoting survival and differentiation ofdendritic cells (MHC II expression, cell size, granularity). Theimmunostimulatory nucleic acids also induce maturation of dendriticcells. Since dendritic cells form the link between the innate and theacquired immune system, by presenting antigens as well as through theirexpression of pattern recognition receptors which detect microbialmolecules like LPS in their local environment, the ability to activatedendritic cells with immunostimulatory nucleic acids supports the use ofthese immunostimulatory nucleic acid based strategies for in vivo andex-vivo immunotherapy against disorders such as cancer and allergic orinfectious diseases. The immunostimulatory nucleic acids are also usefulfor activating and inducing maturation of dendritic cells.

Immunostimulatory nucleic acids also increase natural killer cell lyticactivity and antibody dependent cellular cytotoxicity (ADCC). ADCC canbe performed using a immunostimulatory nucleic acid in combination withan antibody specific for a cellular target, such as a cancer cell. Whenthe immunostimulatory nucleic acid is administered to a subject inconjunction with the antibody the subject's immune system is induced tokill the tumor cell. The antibodies useful in the ADCC procedure includeantibodies which interact with a cell in the body. Many such antibodiesspecific for cellular targets have been described in the art and manyare commercially available. Examples of these antibodies are listedbelow among the list of cancer immunotherapies.

The nucleic acids are also useful for redirecting an immune responsefrom a Th2 immune response to a Th1 immune response. Redirection of animmune response from a Th2 to a Th1 immune response can be assessed bymeasuring the levels of cytokines produced in response to the nucleicacid (e.g., by inducing monocytic cells and other cells to produce Th1cytokines, including IL-12, IFN-γ and GM-CSF). The redirection orrebalance of the immune response from a Th2 to a Th1 response isparticularly useful for the treatment or prevention of asthma. Forinstance, an effective amount for treating asthma can be that amount;useful for redirecting a Th2 type of immune response that is associatedwith asthma to a Th1 type of response. Th2 cytokines, especially IL-4and IL-5 are elevated in the airways of asthmatic subjects. Thesecytokines promote important aspects of the asthmatic inflammatoryresponse, including IgE isotype switching, eosinophil chemotaxis andactivation and mast cell growth. Th1 cytokines, especially IFN-γ andIL-12, can suppress the formation of Th2 clones and production of Th2cytokines. The immunostimulatory nucleic acids of the invention cause anincrease in Th1 cytokines which helps to rebalance the immune system,preventing or reducing the adverse effects associated with apredominately Th2 immune response.

The invention also includes a method for inducing antigen non-specificinnate immune activation and broad spectrum resistance to infectiouschallenge using the immunostimulatory nucleic acids. The term antigennon-specific innate immune activation as used herein refers to theactivation of immune cells other than B cells and for instance caninclude the activation of NK cells, T cells or other immune cells thatcan respond in an antigen independent fashion or some combination ofthese cells. A broad spectrum resistance to infectious challenge isinduced because the immune cells are in active form and are primed torespond to any invading compound or microorganism. The cells do not haveto be specifically primed against a particular antigen. This isparticularly useful in biowarfare, and the other circumstances describedabove such as travelers.

The nucleic acids of the invention can be used in combination with othertherapeutic agents including anti-microbial agents, adjuvants,cytokines, anti-cancer therapies, allergy medicaments, asthmamedicaments, and the like.

The nucleic acids of the invention may be administered to a subject withan anti-microbial agent. An anti-microbial agent, as used herein, refersto a naturally-occurring or synthetic compound which is capable ofkilling or inhibiting infectious microorganisms. The type ofanti-microbial agent useful according to the invention will depend uponthe type of microorganism with which the subject is infected or at riskof becoming infected. Anti-microbial agents include but are not limitedto anti-bacterial agents, anti-viral agents, anti-fungal agents andanti-parasitic agents. Phrases such as “anti-infective agent”,“anti-bacterial agent”, “anti-viral agent”, “anti-fungal agent”,“anti-parasitic agent” and “parasiticide” have well-established meaningsto those of ordinary skill in the art and are defined in standardmedical texts. Briefly, anti-bacterial agents kill or inhibit bacteria,and include antibiotics as well as other synthetic or natural compoundshaving similar functions. Antibiotics are low molecular weight moleculeswhich are produced as secondary metabolites by cells, such asmicroorganisms. In general, antibiotics interfere with one or morebacterial functions or structures which are specific for themicroorganism and which are not present in host cells. Anti-viral agentscan be isolated from natural sources or synthesized and are useful forkilling or inhibiting viruses. Anti-fungal agents are used to treatsuperficial fungal infections as well as opportunistic and primarysystemic fungal infections. Anti-parasite agents kill or inhibitparasites.

Antibacterial agents kill or inhibit the growth or function of bacteria.A large class of antibacterial agents is antibiotics. Antibiotics, whichare effective for killing or inhibiting a wide range of bacteria, arereferred to as broad spectrum antibiotics. Other types of antibioticsare predominantly effective against the bacteria of the classgram-positive or gram-negative. These types of antibiotics are referredto as narrow spectrum antibiotics. Other antibiotics which are effectiveagainst a single organism or disease and not against other types ofbacteria, are referred to as limited spectrum antibiotics. Antibacterialagents are sometimes classified based on their primary mode of action.In general, antibacterial agents are cell wall synthesis inhibitors,cell membrane inhibitors, protein synthesis inhibitors, nucleic acidsynthesis or functional inhibitors, and competitive inhibitors.

Anti-bacterial agents useful in the invention include but are notlimited to natural penicillins, semi-synthetic penicillins, clavulanicacid, cephalolsporins, bacitracin, ampicillin, carbenicillin, oxacillin,azlocillin, mezlocillin, piperacillin, methicillin, dicloxacillin,nafcillin, cephalothin, cephapirin, cephalexin, cefamandole, cefaclor,cefazolin, cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet,cefixime, ceftriaxone, cefoperazone, ceftazidine, moxalactam,carbapenems, imipenems, monobactems, euztreonam, vancomycin, polymyxin,amphotericin B, nystatin, imidazoles, clotrimazole, miconazole,ketoconazole, itraconazole, fluconazole, rifampins, ethambutol,tetracyclines, chloramphenicol, macrolides, aminoglycosides,streptomycin, kanamycin, tobramycin, amikacin, gentamicin, tetracycline,minocycline, doxycycline, chlortetracycline, erythromycin,roxithromycin, clarithromycin, oleandomycin, azithromycin,chloramphenicol, quinolones, co-trimoxazole, norfloxacin, ciprofloxacin,enoxacin, nalidixic acid, temafloxacin, sulfonamides, gantrisin, andtrimethoprim; Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine;Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; AmifloxacinMesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid;Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; AmpicillinSodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate;Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium;Bacampicillin Hydrochloride; Bacitracin; Bacitracin MethyleneDisalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium;Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; BiphenamineHydrochloride; Bispyrithione Magsulfex; Butikacin; Butirosin Sulfate;Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; CarbenicillinIndanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium;Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate;Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium;Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; CefepimeHydrochloride; Cefetecol; Cefixime; Cefinenoxime Hydrochloride;Cefimetazole; Cefimetazole Sodium; Cefonicid Monosodium; CefonicidSodium; Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan;Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium;Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium;Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine;Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium;Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; CephalexinHydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium;Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol;Chloramphenicol; Chloramphenicol Palmitate; Chloramphenicol PantothenateComplex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate;Chloroxylenol; Chlortetracycline Bisulfate; ChlortetracyclineHydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride;Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; CloxacillinSodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin;Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone;Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline;Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium;Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline;Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; DroxacinSodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride;Erythromycin; Erythromycin Acistrate; Erythromycin Estolate;Erythromycin Ethylsuccinate; Erythromycin Gluceptate; ErythromycinLactobionate; Erythromycin Propionate; Erythromycin Stearate; EthambutolHydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid;Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin;Hetacillin Potassium; Hexedine; Ibafloxacin; Imipenem; Isoconazole;Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin;Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin;Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; MeclocyclineSulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem;Methacycline; Methacycline Hydrochloride; Methenamine; MethenamineHippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim;Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin;Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; MirincamycinHydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; NalidixateSodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate;Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate;Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone;Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole;Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium;Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; OxytetracyclineHydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin;Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin GPotassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V;Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin VPotassium; Pentizidone Sodium; Phenyl Aminosalicylate; PiperacillinSodium; Pirbenicillin Sodium; Piridicillin Sodium; PirlimycinHydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin;Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin;Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin;Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin;Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; RosaramicinButyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate;Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline;Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin;Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride;Spiramycin; Stallimycin Hydrochloride; Steffimycin; StreptomycinSulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium;Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine;Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole;Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole;Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl;Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; SuncillinSodium; Talampicillin Hydrochloride; Teicoplanin; TemafloxacinHydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride;Tetracycline Phosphate Complex; Tetroxoprirn; Thiamphenicol;Thiphencillin Potassium; Ticarcillin Cresyl Sodium; TicarcillinDisodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride;Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; TrimethoprimSulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin; andZorbamycin.

Antiviral agents are compounds which prevent infection of cells byviruses or replication of the virus within the cell. There are manyfewer antiviral drugs than antibacterial drugs because the process ofviral replication is so closely related to DNA replication within thehost cell, that non-specific antiviral agents would often be toxic tothe host. There are several stages within the process of viral infectionwhich can be blocked or inhibited by antiviral agents. These stagesinclude, attachment of the virus to the host cell (immunoglobulin orbinding peptides), uncoating of the virus (e.g. amantadine), synthesisor translation of viral mRNA (e.g. interferon), replication of viral RNAor DNA (e.g. nucleoside analogues), maturation of new virus proteins(e.g. protease inhibitors), and budding and release of the virus.

Nucleotide analogues are synthetic compounds which are similar tonucleotides, but which have an incomplete or abnormal deoxyribose orribose group. Once the nucleotide analogues are in the cell, they arephosphorylated, producing the triphosphate formed which competes withnormal nucleotides for incorporation into the viral DNA or RNA. Once thetriphosphate form of the nucleotide analogue is incorporated into thegrowing nucleic acid chain, it causes irreversible association with theviral polymerase and thus chain termination. Nucleotide analoguesinclude, but are not limited to, acyclovir (used for the treatment ofherpes simplex virus and varicella-zoster virus), gancyclovir (usefulfor the treatment of cytomegalovirus), idoxuridine, ribavirin (usefulfor the treatment of respiratory syncitial virus), dideoxyinosine,dideoxycytidine, and zidovudine (azidothymidine).

The interferons are cytokines which are secreted by virus-infected cellsas well as immune cells. The interferons function by binding to specificreceptors on cells adjacent to the infected cells, causing the change inthe cell which protects it from infection by the virus. α andβ-interferon also induce the expression of Class I and Class II MHCmolecules on the surface of infected cells, resulting in increasedantigen presentation for host immune cell recognition. α andβ-interferons are available as recombinant forms and have been used forthe treatment of chronic hepatitis B and C infection. At the dosageswhich are effective for anti-viral therapy, interferons have severe sideeffects such as fever, malaise and weight loss.

Immunoglobulin therapy is used for the prevention of viral infection.Immunoglobulin therapy for viral infections is different than bacterialinfections, because rather than being antigen-specific, theimmunoglobulin therapy functions by binding to extracellular virions andpreventing them from attaching to and entering cells which aresusceptible to the viral infection. The therapy is useful for theprevention of viral infection for the period of time that the antibodiesare present in the host. In general there are two types ofimmunoglobulin therapies, normal immunoglobulin therapy andhyper-immunoglobulin therapy. Normal immune globulin therapy utilizes aantibody product which is prepared from the serum of normal blood donorsand pooled. This pooled product contains low titers of antibody to awide range of human viruses, such as hepatitis A, parvovirus,enterovirus (especially in neonates). Hyper-immune globulin therapyutilizes antibodies which are prepared from the serum of individuals whohave high titers of an antibody to a particular virus. Those antibodiesare then used against a specific virus. Examples of hyper-immuneglobulins include zoster immune globulin (useful for the prevention ofvaricella in immuno-compromised children and neonates), human rabiesimmunoglobulin (useful in the post-exposure prophylaxis of a subjectbitten by a rabid animal), hepatitis B immune globulin (useful in theprevention of hepatitis B virus, especially in a subject exposed to thevirus), and RSV immune globulin (useful in the treatment of respiratorysyncitial virus infections).

Another type of immunoglobulin therapy is active immunization. Thisinvolves the administration of antibodies or antibody fragments to viralsurface proteins. Two types of vaccines which are available for activeimmunization of hepatitis B include serum-derived hepatitis B antibodiesand recombinant hepatitis B antibodies. Both are prepared from HBsAg.The antibodies are administered in three doses to subjects at high riskof infection with hepatitis B virus, such as health care workers, sexualpartners of chronic carriers, and infants.

Thus, anti-viral agents useful in the invention include but are notlimited to immunoglobulins, amantadine, interferon, nucleosideanalogues, and protease inhibitors. Specific examples of anti-viralsinclude but are not limited to Acemannan; Acyclovir; Acyclovir Sodium;Adefovir; Alovudine; Alvircept Sudotox; Amantadine Hydrochloride;Aranotin; Arildone; Atevirdine Mesylate; Avridine; Cidofovir;Cipamfylline; Cytarabine Hydrochloride; Delavirdine Mesylate;Desciclovir; Didanosine; Disoxaril; Edoxudine; Enviradene; Enviroxime;Famciclovir; Famotine Hydrochloride; Fiacitabine; Fialuridine;Fosarilate; Foscarnet Sodium; Fosfonet Sodium; Ganciclovir; GanciclovirSodium; Idoxuridine; Kethoxal; Lamivudine; Lobucavir; MemotineHydrochloride; Methisazone; Nevirapine; Penciclovir; Pirodavir;Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate; SomantadineHydrochloride; Sorivudine; Statolon; Stavudine; Tilorone Hydrochloride;Trifluridine; Valacyclovir Hydrochloride; Vidarabine; VidarabinePhosphate; Vidarabine Sodium Phosphate; Viroxime; Zalcitabine;Zidovudine; and Zinviroxime.

Anti-fungal agents are useful for the treatment and prevention ofinfective fungi. Anti-fungal agents are sometimes classified by theirmechanism of action. Some anti-fungal agents function as cell wallinhibitors by inhibiting glucose synthase. These include, but are notlimited to, basiungin/ECB. Other anti-fungal agents function bydestabilizing membrane integrity. These include, but are not limited to,immidazoles, such as clotrimazole, sertaconzole, fluconazole,itraconazole, ketoconazole, miconazole, and voriconacole, as well as FK463, amphotericin B, BAY 38-9502, MK 991, pradimicin, UK 292,butenafine, and terbinafine. Other anti-fungal agents function bybreaking down chitin (e.g. chitinase) or immunosuppression (501 cream).Some examples of commercially-available agents are shown in Table 1.

TABLE 1 Company Brand Name Generic Name Indication Mechanism of ActionPHARMACIA & PNU 196443 PNU 196443 Anti Fungal n/k UP JOHN Lilly LY303366 Basiungin/ECB Fungal Infections Anti-fungal/cell wall inhibitor,glucose synthase inhibitor Bayer Canesten Clotrimazole Fungal InfectionsMembrane integrity destabilizer Fujisawa FK 463 FK 463 Fungal InfectionsMembrane integrity destabilizer Mylan Sertaconzaole Sertaconzole FungalInfections Membrane integrity destabilizer Genzyme Chitinase ChitinaseFungal Infections, Systemic Chitin Breakdown Liposome AbelcetAmphotericin B, Fungal Infections, Systemic Membrane integritydestabilizer Liposomal Sequus Amphotec Amphotericin B, FungalInfections, Systemic Membrane integrity destabilizer Liposomal Bayer BAY38-9502 BAY 38-9502 Fungal Infections, Systemic Membrane integritydestabilizer Pfizer Diflucan Fluconazole Fungal Infections, SystemicMembrane integrity destabilizer Johnson & Sporanox Itraconazole FungalInfections, Systemic Membrane integrity destabilizer Johnson SepracorItraconzole (2R, Itraconzole (2R, 4S) Fungal Infections, SystemicMembrane integrity destabilizer 4S) Johnson & Nizoral KetoconazoleFungal Infections, Systemic Membrane integrity destabilizer JohnsonJohnson & Monistat Miconazole Fungal Infections, Systemic Membraneintegrity destabilizer Johnson Merck MK 991 MK 991 Fungal Infections,Systemic Membrane integrity destabilizer Bristol Myers PradimicinPradimicin Fungal Infections, Systemic Membrane integrity destabilizerSq'b Pfizer UK-292, 663 UK-292, 663 Fungal Infections, Systemic Membraneintegrity destabilizer Pfizer Voriconazole Voriconazole FungalInfections, Systemic Membrane integrity destabilizer Mylan 501 Cream 501Cream Inflammatory Fungal Immunosuppression Conditions Mylan MentaxButenafine Nail Fungus Membrane Integrity Destabiliser Schering PloughAnti Fungal Anti Fungal Opportunistic Infections Membrane IntegrityDestabiliser Alza Mycelex Troche Clotrimazole Oral Thrush MembraneIntegrity Stabliser Novartis Lamisil Terbinafine Systemic FungalInfections, Membrane Integrity Destabiliser Onychomycosis

Thus, the anti-fungal agents useful in the invention include but are notlimited to imidazoles, FK 463, amphotericin B, BAY 38-9502, MK 991,pradimicin, UK 292, butenafine, chitinase, 501 cream, Acrisorcin;Ambruticin; Amorolfine, Amphotericin B; Azaconazole; Azaserine;Basifungin; Bifonazole; Biphenamine Hydrochloride; BispyrithioneMagsulfex; Butoconazole Nitrate; Calcium Undecylenate; Candicidin;Carbol-Fuchsin; Chlordantoin; Ciclopirox; Ciclopirox Olamine;Cilofungin; Cisconazole; Clotrimazole; Cuprimyxin; Denofungin;Dipyrithione; Doconazole; Econazole; Econazole Nitrate; Enilconazole;Ethonam Nitrate; Fenticonazole Nitrate; Filipin; Fluconazole;Flucytosine; Fungimycin; Griseofulvin; Hamycin; Isoconazole;Itraconazole; Kalafungin; Ketoconazole; Lomofungin; Lydimycin;Mepartricin; Miconazole; Miconazole Nitrate; Monensin; Monensin Sodium;Naftifine Hydrochloride; Neomycin Undecylenate; Nifuratel; Nifurmerone;Nitralamine Hydrochloride; Nystatin; Octanoic Acid; Orconazole Nitrate;Oxiconazole Nitrate; Oxifungin Hydrochloride; Parconazole Hydrochloride;Partricin; Potassium Iodide; Proclonol; Pyrithione Zinc; Pyrrolnitrin;Rutamycin; Sanguinarium Chloride; Saperconazole; Scopafungin; SeleniumSulfide; Sinefungin; Sulconazole Nitrate; Terbinafine; Terconazole;Thiram; Ticlatone; Tioconazole; Tolciclate; Tolindate; Tolnaftate;Triacetin; Triafungin; Undecylenic Acid; Viridofulvin; ZincUndecylenate; and Zinoconazole Hydrochloride.

Examples of anti-parasitic agents, also referred to as parasiticidesuseful for human administration include but are not limited toalbendazole, amphotericin B, benznidazole, bithionol, chloroquine HCl,chloroquine phosphate, clindamycin, dehydroemetine, diethylcarbamazine,diloxamide furoate, eflornithine, furazolidaone, glucocorticoids,halofantrine, iodoquinol, ivermectin, mebendazole, mefloquine, meglumineantimoniate, melarsoprol, metrifonate, metronidazole, niclosamide,nifurtimox, oxamniquine, paromomycin, pentamidine isethionate,piperazine, praziquantel, primaquine phosphate, proguanil, pyrantelpamoate, pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine,quinacrine HCl, quinine sulfate, quinidine gluconate, spiramycin,stibogluconate sodium (sodium antimony gluconate), suramin,tetracycline, doxycycline, thiabendazole, tinidazole,trimethroprim-sulfamethoxazole, and tryparsamide some of which are usedalone or in combination with others.

Parasiticides used in non-human subjects include piperazine,diethylcarbamazine, thiabendazole, fenbendazole, albendazole,oxfendazole, oxibendazole, febantel, levamisole, pyrantel tartrate,pyrantel pamoate, dichlorvos, ivermectin, doramectic, milbemycin oxime,iprinomectin, moxidectin, N-butyl chloride, toluene, hygromycin Bthiacetarsemide sodium, melarsomine, praziquantel, epsiprantel,benzimidazoles such as fenbendazole, albendazole, oxfendazole,clorsulon, albendazole, amprolium; decoquinate, lasalocid, monensinsulfadimethoxine; sulfamethazine, sulfaquinoxaline, metronidazole.

Parasiticides used in horses include mebendazole, oxfendazole, febantel,pyrantel, dichlorvos, trichlorfon, ivermectin, piperazine; for S.westeri: ivermectin, benzimiddazoles such as thiabendazole,cambendazole, oxibendazole and fenbendazole. Useful parasiticides indogs include milbemycin oxine, ivermectin, pyrantel pamoate and thecombination of ivermectin and pyrantel. The treatment of parasites inswine can include the use of levamisole, piperazine, pyrantel,thiabendazole, dichlorvos and fenbendazole. In sheep and goatsanthelmintic agents include levamisole or ivermectin. Caparsolate hasshown some efficacy in the treatment of D. immitis (heartworm) in cats.

The immunostimulatory nucleic acids may also be administered inconjunction with an anti-cancer therapy. Anti-cancer therapies includecancer medicaments, radiation and surgical procedures. As used herein, a“cancer medicament” refers to a agent which is administered to a subjectfor the purpose of treating a cancer. As used herein, “treating cancer”includes preventing the development of a cancer, reducing the symptomsof cancer, and/or inhibiting the growth of an established cancer. Inother aspects, the cancer medicament is administered to a subject atrisk of developing a cancer for the purpose of reducing the risk ofdeveloping the cancer. Various types of medicaments for the treatment ofcancer are described herein. For the purpose of this specification,cancer medicaments are classified as chemotherapeutic agents,immunotherapeutic agents, cancer vaccines, hormone therapy, andbiological response modifiers.

As used herein, a “cancer medicament” refers to an agent which isadministered to a subject for the purpose of treating a cancer. As usedherein, “treating cancer” includes preventing the development of acancer, reducing the symptoms of cancer, and/or inhibiting the growth ofan established cancer. In other aspects, the cancer medicament isadministered to a subject at risk of developing a cancer for the purposeof reducing the risk of developing the cancer. Various types ofmedicaments for the treatment of cancer are described herein. For thepurpose of this specification, cancer medicaments are classified aschemotherapeutic agents, immunotherapeutic agents, cancer vaccines,hormone therapy, and biological response modifiers. Additionally, themethods of the invention are intended to embrace the use of more thanone cancer medicament along with the immunostimulatory nucleic acids. Asan example, where appropriate, the immunostimulatory nucleic acids maybe administered with a both a chemotherapeutic agent and animmunotherapeutic agent. Alternatively, the cancer medicament mayembrace an immunotherapeutic agent and a cancer vaccine, or achemotherapeutic agent and a cancer vaccine, or a chemotherapeuticagent, an immunotherapeutic agent and a cancer vaccine all administeredto one subject for the purpose of treating a subject having a cancer orat risk of developing a cancer.

Cancer medicaments function in a variety of ways. Some cancermedicaments work by targeting physiological mechanisms that are specificto tumor cells. Examples include the targeting of specific genes andtheir gene products (i.e., proteins primarily) which are mutated incancers. Such genes include but are not limited to oncogenes (e.g., Ras,Her2, bcl-2), tumor suppressor genes (e.g., EGF, p53, Rb), and cellcycle targets (e.g., CDK4, p21, telomerase). Cancer medicaments canalternately target signal transduction pathways and molecular mechanismswhich are altered in cancer cells. Targeting of cancer cells via theepitopes expressed on their cell surface is accomplished through the useof monoclonal antibodies. This latter type of cancer medicament isgenerally referred to herein as immunotherapy.

Other cancer medicaments target cells other than cancer cells. Forexample, some medicaments prime the immune system to attack tumor cells(i.e., cancer vaccines). Still other medicaments, called angiogenesisinhibitors, function by attacking the blood supply of solid tumors.Since the most malignant cancers are able to metastasize (i.e., existthe primary tumor site and seed a distal tissue, thereby forming asecondary tumor), medicaments that impede this metastasis are alsouseful in the treatment of cancer. Angiogenic mediators include basicFGF, VEGF, angiopoietins, angiostatin, endostatin, TNFα, TNP-470,thrombospondin-1, platelet factor 4, CAI, and certain members of theintegrin family of proteins. One category of this type of medicament isa metalloproteinase inhibitor, which inhibits the enzymes used by thecancer cells to exist the primary tumor site and extravasate intoanother tissue.

Immunotherapeutic agents are medicaments which derive from antibodies orantibody fragments which specifically bind or recognize a cancerantigen. As used herein a cancer antigen is broadly defined as anantigen expressed by a cancer cell. Preferably, the antigen is expressedat the cell surface of the cancer cell. Even more preferably, theantigen is one which is not expressed by normal cells, or at least notexpressed to the same level as in cancer cells. Antibody-basedimmunotherapies may function by binding to the cell surface of a cancercell and thereby stimulate the endogenous immune system to attack thecancer cell. Another way in which antibody-based therapy functions is asa delivery system for the specific targeting of toxic substances tocancer cells. Antibodies are usually conjugated to toxins such as ricin(e.g., from castor beans), calicheamicin and maytansinoids, toradioactive isotopes such as Iodine-131 and Yttrium-90, tochemotherapeutic agents (as described herein), or to biological responsemodifiers. In this way, the toxic substances can be concentrated in theregion of the cancer and non-specific toxicity to normal cells can beminimized. In addition to the use of antibodies which are specific forcancer antigens, antibodies which bind to vasculature, such as thosewhich bind to endothelial cells, are also useful in the invention. Thisis because generally solid tumors are dependent upon newly formed bloodvessels to survive, and thus most tumors are capable of recruiting andstimulating the growth of new blood vessels. As a result, one strategyof many cancer medicaments is to attack the blood vessels feeding atumor and/or the connective tissues (or stroma) supporting such bloodvessels.

The use of immunostimulatory nucleic acids in conjunction withimmunotherapeutic agents such as monoclonal antibodies is able toincrease long-term survival through a number of mechanisms includingsignificant enhancement of ADCC (as discussed above), activation ofnatural killer (NK) cells and an increase in IFNα levels. The nucleicacids when used in combination with monoclonal antibodies serve toreduce the dose of the antibody required to achieve a biological result.

Examples of cancer immunotherapies which are currently being used orwhich are in development are listed in Table 2.

TABLE 2 Cancer Immunotherapies in Development or on the Market MARKETERBRAND NAME (GENERIC NAME) INDICATION IDEC/Genentech, Rituxan ™(rituximab, Mabthera) (IDEC- non-Hodgkin's Inc./Hoffmann-LaRoche C2B8,chimeric murine/human anti-CD20 lymphoma (first monoclonal antibody MAb)licensed for the treatment of cancer in the U.S.)Genentech/Hoffmann-LaRoche Herceptin, anti-Her2 hMAb Breast/ovarianCytogen Corp. Quadramet (CYT-424) radiotherapeutic Bone metastases agentCentocor/Glaxo/Ajinomoto Panorex ® (17-1A) (murine monoclonal Adjuvanttherapy for antibody) colorectal (Dukes-C) Centocor/Ajinomoto Panorex ®(17-1A) (chimeric murine Pancreatic, lung, monoclonal antibody) breast,ovary IDEC IDEC-Y2B8 (murine, anti-CD20 MAb non-Hodgkin's labeled withYttrium-90) lymhoma ImClone Systems BEC2 (anti-idiotypic MAb, mimics theSmall cell lung GD₃ epitope) (with BCG) ImClone Systems C225 (chimericmonoclonal antibody to Renal cell epidermal growth factor receptor(EGFr)) Techniclone Oncolym (Lym-1 monoclonal antibody non-Hodgkin'sInternational/Alpha linked to 131 iodine) lymphoma Therapeutics ProteinDesign Labs SMART M195 Ab, humanized Acute myleoid leukemia Techniclone¹³¹I LYM-1 (Oncolym ™) non-Hodgkin's Corporation/Cambridge lymphomaAntibody Technology Aronex Pharmaceuticals, ATRAGEN ® Acutepromyelocytic Inc. leukemia ImClone Systems C225 (chimeric anti-EGFrmonoclonal Head & neck, non- antibody) + cisplatin or radiation smallcell lung cancer Altarex, Canada Ovarex (B43.13, anti-idiotypic CA125,Ovarian mouse MAb) Coulter Pharma (Clinical Bexxar (anti-CD20 Mablabeled with ¹³¹I) non-Hodgkin's results have been positive, lymphomabut the drug has been associated with significant bone marrow toxicity)Aronex Pharmaceuticals, ATRAGEN ® Kaposi's sarcoma Inc. IDECPharmaceuticals Rituxan ™ (MAb against CD20) pan-B Ab B cell lymphomaCorp./Genentech in combo. with chemotherapy LeukoSite/Ilex OncologyLDP-03, huMAb to the leukocyte antigen Chronic lymphocytic CAMPATHleukemia (CLL) Center of Molecular ior t6 (anti CD6, murine MAb) CTCLCancer Immunology Medarex/Novartis MDX-210 (humanized anti-HER-2 Breast,ovarian bispecific antibody) Medarex/Novartis MDX-210 (humanizedanti-HER-2 Prostate, non-small cell bispecific antibody) lung,pancreatic, breast Medarex MDX-11 (complement activating receptor Acutemyelogenous (CAR) monoclonal antibody) leukemia (AML) Medarex/NovartisMDX-210 (humanized anti-HER-2 Renal and colon bispecific antibody)Medarex MDX-11 (complement activating receptor Ex vivo bone marrow (CAR)monoclonal antibody) purging in acute myelogenous leukemia (AML) MedarexMDX-22 (humanized bispecific antibody, Acute myleoid MAb-conjugates)(complement cascade leukemia activators) Cytogen OV103 (Yttrium-90labelled antibody) Ovarian Cytogen OV103 (Yttrium-90 labelled antibody)Prostate Aronex Pharmaceuticals, ATRAGEN ® non-Hodgkin's Inc. lymphomaGlaxo Wellcome plc 3622W94 MAb that binds to EGP40 (17- non-small celllung, 1A) pancarcinoma antigen on prostate (adjuvant) adenocarcinomasGenentech Anti-VEGF, RhuMAb (inhibits Lung, breast, prostate,angiogenesis) colorectal Protein Design Labs Zenapax (SMART Anti-Tac(IL-2 Leukemia, lymphoma receptor) Ab, humanized) Protein Design LabsSMART M195 Ab, humanized Acute promyelocytic leukemia ImClone SystemsC225 (chimeric anti-EGFr monoclonal Breast antibody) + taxol ImCloneSystems (licensed C225 (chimeric anti-EGFr monoclonal prostate from RPR)antibody) + doxorubicin ImClone Systems C225 (chimeric anti-EGFrmonoclonal prostate antibody) + adriamycin ImClone Systems BEC2(anti-idiotypic MAb, mimics the Melanoma GD₃ epitope) Medarex MDX-210(humanized anti-HER-2 Cancer bispecific antibody) Medarex MDX-220(bispecific for tumors that Lung, colon, prostate, express TAG-72)ovarian, endometrial, pancreatic and gastric Medarex/Novartis MDX-210(humanized anti-HER-2 Prostate bispecific antibody) Medarex/Merck KgaAMDX-447 (humanized anti-EGF receptor EGF receptor cancers bispecificantibody) (head & neck, prostate, lung, bladder, cervical, ovarian)Medarex/Novartis MDX-210 (humanized anti-HER-2 Comb. Therapy withbispecific antibody) G-CSF for various cancers, esp. breast IDECMELIMMUNE-2 (murine monoclonal Melanoma antibody therapeutic vaccine)IDEC MELIMMUNE-1 (murine monoclonal Melanoma antibody therapeuticvaccine) Immunomedics, Inc. CEACIDE ™ (I-131) Colorectal and other NeoRxPretarget ™ radioactive antibodies non-Hodgkin's B cell lymphomaNovopharm Biotech, Inc. NovoMAb-G2 (pancarcinoma specific Cancer Ab)Techniclone Corporation/ TNT (chimeric MAb to histone antigens) BrainCambridge Antibody Technology Techniclone International/ TNT (chimericMAb to histone antigens) Brain Cambridge Antibody Technology NovopharmGliomab-H (Monoclonals - Humanized Brain, melanomas, Abs) neuroblastomasGenetics Institute/AHP GNI-250 Mab Colorectal Merck KgaA EMD-72000(chimeric-EGF antagonist) Cancer Immunomedics LymphoCide (humanized LL2antibody) non-Hodgkin's B-cell lymphoma Immunex/AHP CMA 676 (monoclonalantibody Acute myelogenous conjugate) leukemia Novopharm Biotech, Inc.Monopharm-C Colon, lung, pancreatic Novopharm Biotech, Inc. 4B5anti-idiotype Ab Melanoma, small-cell lung Center of Molecular ioregf/r3 (anti EGF-R humanized Ab) Radioimmunotherapy Immunology Center ofMolecular ior c5 (murine MAb colorectal) for Colorectal Immunologyradioimmunotherapy Creative BioMolecules/ BABS (biosynthetic antibodybinding site) Breast cancer Chiron Proteins ImClone Systems/Chugai FLK-2(monoclonal antibody to fetal liver Tumor-associated kinase-2 (FLK-2))angiogenesis ImmunoGen, Inc. Humanized MAb/small-drug conjugateSmall-cell lung Medarex, Inc. MDX-260 bispecific, targets GD-2 Melanoma,glioma, neuroblastoma Procyon Biopharma, Inc. ANA Ab Cancer ProteinDesign Labs SMART 1D10 Ab B-cell lymphoma Protein Design SMART ABL 364Ab Breast, lung, colon Labs/Novartis Immunomedics, Inc. ImmuRAIT-CEAColorectal

Yet other types of chemotherapeutic agents which can be used accordingto the invention include Aminoglutethimide, Asparaginase, Busulfan,Carboplatin, Chlorombucil, Cytarabine HCI, Dactinomycin, DaunorubicinHCl, Estramustine phosphate sodium, Etoposide (VP16-213), Floxuridine,Fluorouracil (5-FU), Flutamide, Hydroxyurea (hydroxycarbamide),Ifosfamide, Interferon Alfa-2a, Alfa-2b, Leuprolide acetate(LHRH-releasing factor analogue), Lomustine (CCNU), Mechlorethamine HCl(nitrogen mustard), Mercaptopurine, Mesna, Mitotane (o.p′-DDD),Mitoxantrone HCl, Octreotide, Plicamycin, Procarbazine HCl,Streptozocin, Tamoxifen citrate, Thioguanine, Thiotepa, Vinblastinesulfate, Amsacrine (m-AMSA), Azacitidine, Erthropoietin,Hexamethylmelamine (HMM), Interleukin 2, Mitoguazone (methyl-GAG; methylglyoxal bis-guanylhydrazone; MGBG), Pentostatin (2′deoxycoformycin),Semustine (methyl-CCNU), Teniposide (VM-26) and Vindesine sulfate.

Cancer vaccines are medicaments which are intended to stimulate anendogenous immune response against cancer cells. Currently producedvaccines predominantly activate the humoral immune system (i.e., theantibody dependent immune response). Other vaccines currently indevelopment are focused on activating the cell-mediated immune systemincluding cytotoxic T lymphocytes which are capable of killing tumorcells. Cancer vaccines generally enhance the presentation of cancerantigens to both antigen presenting cells (e.g., macrophages anddendritic cells) and/or to other immune cells such as T cells, B cells,and NK cells.

Although cancer vaccines may take one of several forms, as discussedinfra, their purpose is to deliver cancer antigens and/or cancerassociated antigens to antigen presenting cells (APC) in order tofacilitate the endogenous processing of such antigens by APC and theultimate presentation of antigen presentation on the cell surface in thecontext of MHC class I molecules. One form of cancer vaccine is a wholecell vaccine which is a preparation of cancer cells which have beenremoved from a subject, treated ex vivo and then reintroduced as wholecells in the subject. Lysates of tumor cells can also be used as cancervaccines to elicit an immune response. Another form cancer vaccine is apeptide vaccine which uses cancer-specific or cancer-associated smallproteins to activate T cells. Cancer-associated proteins are proteinswhich are not exclusively expressed by cancer cells (i.e., other normalcells may still express these antigens). However, the expression ofcancer-associated antigens is generally consistently upregulated withcancers of a particular type. Yet another form of cancer vaccine is adendritic cell vaccine which includes whole dendritic cells which havebeen exposed to a cancer antigen or a cancer-associated antigen invitro. Lysates or membrane fractions of dendritic cells may also be usedas cancer vaccines. Dendritic cell vaccines are able to activateantigen-presenting cells directly. Other cancer vaccines includeganglioside vaccines, heat-shock protein vaccines, viral and bacterialvaccines, and nucleic acid vaccines.

The use of immunostimulatory nucleic acids in conjunction with cancervaccines provides an improved antigen-specific humoral and cell mediatedimmune response, in addition to activating NK cells and endogenousdendritic cells, and increasing IFNα levels. This enhancement allows avaccine with a reduced antigen dose to be used to achieve the samebeneficial effect. In some instances, cancer vaccines may be used alongwith adjuvants, such as those described above.

Other vaccines take the form of dendritic cells which have been exposedto cancer antigens in vitro, have processed the antigens and are able toexpress the cancer antigens at their cell surface in the context of MHCmolecules for effective antigen presentation to other immune systemcells.

The immunostimulatory nucleic acids are used in one aspect of theinvention in conjunction with cancer vaccines which are dendritic cellbased. A dendritic cell is a professional antigen presenting cell.Dendritic cells form the link between the innate and the acquired immunesystem by presenting antigens and through their expression of patternrecognition receptors which detect microbial molecules like LPS in theirlocal environment. Dendritic cells efficiently internalize, process, andpresent soluble specific antigen to which it is exposed. The process ofinternalizing and presenting antigen causes rapid upregulation of theexpression of major histocompatibility complex (MHC) and costimulatorymolecules, the production of cytokines, and migration toward lymphaticorgans where they are believed to be involved in the activation of Tcells.

Table 3 lists a variety of cancer vaccines which are either currentlybeing used or are in development.

TABLE 3 Cancer Vaccines in Development or on the Market BRAND NAME(GENERIC MARKETER NAME) INDICATION Center of Molecular EGF CancerImmunology Center of Molecular Ganglioside cancer Immunology vaccineCenter of Molecular Anti-idiotypic Cancer vaccine Immunology ImCloneSystems/Memorial Gp75 antigen Melanoma Sloan-Kettering Cancer CenterImClone Systems/Memorial Anti-idiotypic Abs Cancer vaccinesSloan-Kettering Cancer Center Progenics Pharmaceuticals, Inc. GMKmelanoma vaccine Melanoma Progenics Pharmaceuticals, Inc, MGVganglioside conjugate Lymphoma, colorectal, vaccine lung Corixa Her2/neuBreast, ovarian AltaRex Ovarex Ovarian AVAX Technologies Inc. M-Vax,autologous whole cell Melanoma AVAX Technologies Inc. O-Vax, autologouswhole cell Ovarian AVAX Technologies Inc. L-Vax, autologous whole cellLeukemia-AML Biomira Inc./Chiron Theratope, STn-KLH Breast, ColorectalBiomira Inc. BLP25, MUC-1 peptide vaccine Lung encapsulated in liposomaldelivery system Biomira Inc. BLP25, MUC-1 peptide vaccine Lungencapsulated in liposomal delivery system + Liposomal IL-2 Biomira Inc.Liposomal idiotypic vaccine Lymphoma B-cell malignancies Ribi ImmunochemMelacine, cell lysate Melanoma Corixa Peptide antigens, microsphereBreast delivery sysem and LeIF adjuvant Corixa Peptide antigens,microsphere Prostate delivery sysem and LeIF adjuvant Corixa Peptideantigens, microsphere Ovarian delivery sysem and LeIF adjuvant CorixaPeptide antigens, microsphere Lymphoma delivery sysem and LeIF adjuvantCorixa Peptide antigens, microsphere Lung delivery sysem and LeIFadjuvant Virus Research Institute Toxin/antigen recombinant All cancersdelivery system Apollon Inc. Genevax-TCR T-cell lymphoma Bavarian NordicResearch MVA-based (vaccinia virus) Melanoma Institute A/S vaccineBioChem Pharma/BioChem PACIS, BCG vaccine Bladder Vaccine CantabPharmaceuticals TA-HPV Cervical Cantab Pharmaceuticals TA-CIN CervicalCantab Pharmaceuticals DISC-Virus, immunotherapy Cancer Pasteur MerieuxConnaught ImmuCyst ®/TheraCys ® - BCG Bladder Immunotherapeutic(Bacillus Calmette-Guerin/Connaught), for intravesical treatment ofsuperficial bladder cancer

As used herein, chemotherapeutic agents embrace all other forms ofcancer medicaments which do not fall into the categories ofimmunotherapeutic agents or cancer vaccines. Chemotherapeutic agents asused herein encompass both chemical and biological agents. These agentsfunction to inhibit a cellular activity which the cancer cell isdependent upon for continued survival. Categories of chemotherapeuticagents include alkylating/alkaloid agents, antimetabolites, hormones orhormone analogs, and miscellaneous antineoplastic drugs. Most if not allof these agents are directly toxic to cancer cells and do not requireimmune stimulation. Combination chemotherapy and immunostimulatorynucleic acid administration increases the maximum tolerable dose ofchemotherapy.

Chemotherapeutic agents which are currently in development or in use ina clinical setting are shown in Table 4.

TABLE 4 Cancer Drugs in Development or on the Market Marketer Brand NameGeneric Name Indication Abbott TNP 470/AGM 1470 FragylineAnti-Angiogenesis in Cancer Takeda TNP 470/AGM 1470 FragylineAnti-Angiogenesis in Cancer Scotia Meglamine GLA Meglamine GLA BladderCancer Medeva Valstar Valrubicin Bladder Cancer - Refractory in situcarcinoma Medeva Valstar Valrubicin Bladder Cancer - Papillary CancerRhone Poulenc Gliadel Wafer Carmustaine + Polifepr Brain Tumor OsanWarner Lambert Undisclosed Cancer (b) Undisclosed Cancer (b) CancerBristol Myers RAS Famesyl Transferase RAS FamesylTransferase CancerSquib Inhibitor Inhibitor Novartis MMI 270 MMI 270 Cancer Bayer BAY12-9566 BAY 12-9566 Cancer Merck Famesyl Transferase Inhibitor FamesylTransferase Cancer (Solid tumors - Inhibitor pancrease, colon, lung,breast) Pfizer PFE MMP Cancer, angiogenesis Pfizer PFE Tyrosine KinaseCancer, angiogenesis Lilly MTA/LY 231514 MTA/LY 231514 Cancer SolidTumors Lilly LY 264618/Lometexol Lometexol Cancer Solid Tumors ScotiaGlamolec LiGLA (lithium-gamma Cancer, pancreatic, breast, linolenate)colon Warner Lambert CI-994 CI-994 Cancer, Solid Tumors/ LeukemiaSchering AG Angiogenesis inhibitor Angiogenesis Inhibitor Cancer/CardioTakeda TNP-470 n/k Malignant Tumor Smithkline Hycamtin TopotecanMetastatic Ovarian Cancer Beecham Novartis PKC 412 PKC 412 Multi-DrugResistant Cancer Novartis Valspodar PSC 833 Myeloid Leukemia/OvarianCancer Immunex Novantrone Mitoxantrone Pain related to hormonerefractory prostate cancer. Warner Lambert Metaret Suramin ProstateGenentech Anti-VEGF Anti-VEGF Prostate/Breast/ Colorectal/NSCL CancerBritish Biotech Batimastat Batimastat (BB94) Pterygium Eisai E 7070 E7070 Solid Tumors Biochem BCH-4556 BCH-4556 Solid Tumors Pharma SankyoCS-682 CS-682 Solid Tumors Agouron AG2037 AG2037 Solid Tumors IDECPharma 9-AC 9-AC Solid Tumors Agouron VEGF/b-FGF Inhibitors VEGF/b-FGFInhibitors Solid Tumors Agouron AG3340 AG3340 Solid Tumors/Macular DegenVertex Incel VX-710 Solid Tumors - IV Vertex VX-853 VX-853 SolidTumors - Oral Zeneca ZD 0101 (inj) ZD 0101 Solid Tumors Novartis ISI 641ISI 641 Solid Tumors Novartis ODN 698 ODN 698 Solid Tumors TanubeSeiyaku TA 2516 Marimistat Solid Tumors British Biotech MarimastatMarimastat (BR 2516) Solid Tumors Celltech CDP 845 Aggrecanase InhibitorSolid Tumors/Breast Cancer Chiroscience D2163 D2163 SolidTumors/Metastases Warner Lambert PD 183805 PD 183805 Daiichi DX8951fDX8951f Anti-Cancer Daiichi Lemonal DP 2202 Lemonal DP 2202 Anti-CancerFujisawa FK 317 FK 317 Anticancer Antibiotic Chugai Picibanil OK-432Antimalignant Tumor Nycomed AD 32/valrubicin Valrubicin BladderCancer-Refractory Amersham Insitu Carcinoma Nycomed Metastron StrontiumDerivative Bone Cancer (adjunt Amersham therapy, Pain) Schering PloughTemodal Temozolomide Brain Tumours Schering Plough Temodal TemozolonideBrain Tumours Liposome Evacet Doxorubicin, Liposomal Breast CancerNycomed Yewtaxan Paclitaxel Breast Cancer Advanced, Amersham OvarianCancer Advanced Bristol Myers Taxol Paclitaxel Breast Cancer Advanced,Squib Ovarian Cancer Advanced, NSCLC Roche Xeloda Capecitabine BreastCancer, Colorectal Cancer Roche Furtulon Doxifluridine Breast Cancer,Colorectal Cancer, Gastric Cancer Pharmacia & Adriamycin DoxorubicinBreast Cancer, Leukemia Upjohn Ivax Cyclopax Paclitaxel, OralBreast/Ovarian Cancer Rhone Poulenc Oral Taxoid Oral Taxoid Broad CancerAHP Novantrone Mitoxantrone Cancer Sequus SPI-077 Cisplatin, StealthCancer Hoechst HMR 1275 Flavopiridol Cancer Pfizer CP-358, 774 EGFRCancer Pfizer CP-609, 754 RAS Oncogene Inhibitor Cancer Bristol MyersBMS-182751 Oral Platinum Cancer (Lung, Ovarian) Squib Bristol Myers UFT(Tegafur/Uracil) UFT (Tegafur/Uracil) Cancer Oral Squib Johnson &Ergamisol Levamisole Cancer Therapy Johnson Glaxo WellcomeEniluracil/776C85 5FU Enhancer Cancer, Refractory Solid & ColorectalCancer Johnson & Ergamisol Levamisole Colon Cancer Johnson Rhone PoulencCampto Irinotecan Colorectal Cancer, Cervical Cancer Pharmacia &Camptosar Irinotecan Colorectal Cancer, Cervical Upjohn Cancer ZenecaTomudex Ralitrexed Colorectal Cancer, Lung Cancer, Breast Cancer Johnson& Leustain Cladribine Hairy Cell Leukaemia Johnson Ivax PaxenePaclitaxel Kaposi Sarcoma Sequus Doxil Doxorubicin, Liposomal KS/CancerSequus Caelyx Doxorubicin, Liposomal KS/Cancer Schering AG FludaraFludarabine Leukaemia Pharmacia & Pharmorubicin Epirubicin Lung/BreastCancer Upjohn Chiron DepoCyt DepoCyt Neoplastic Meningitis Zeneca ZD1839ZD 1839 Non Small Cell Lung Cancer, Pancreatic Cancer BASF LU 79553Bis-Naphtalimide Oncology BASF LU 103793 Dolastain Oncology SheringPlough Caetyx Doxorubicin-Liposome Ovarian/Breast Cancer Lilly GemzarGemcitabine Pancreatic Cancer, Non Small Cell Lung Cancer, Breast,Bladder and Ovarian Zeneca ZD 0473/Anormed ZD 0473/Anormed Platinumbased NSCL, ovarian etc. Yamanouchi YM 116 YM 116 Prostate CancerNycomed Seeds/I-125 Rapid St Lodine Seeds Prostate Cancer AmershamAgouron Cdk4/cdk2 inhibitors cdk4/cdk2 inhibitors Solid Tumors AgouronPARP inhibitors PARP Inhibitors Solid Tumors Chiroscience D4809Dexifosamide Solid Tumors Bristol Myers UFT (Tegafur/Uracil) UFT(Tegafur/Uracil) Solid Tumors Squib Sankyo Krestin Krestin Solid TumorsAsta Medica Ifex/Mesnex Ifosamide Solid Tumors Bristol MeyersIfex/Mesnex Ifosamide Solid Tumors Squib Bristol Myers Vumon TeniposideSolid Tumors Squib Bristol Myers Paraplatin Carboplatin Solid TumorsSquib Bristol Myers Plantinol Cisplatin, Stealth Solid Tumors SquibBristol Myers Plantinol Cisplatin Solid Tumors Squib Bristol MyersVepeside Etoposide Solid Tumors Melanoma Squib Zeneca ZD 9331 ZD 9331Solid Tumors, Advanced Colorectal Chugai Taxotere Docetaxel SolidTumors, Breast Cancer Rhone Poulenc Taxotere Docetaxel Solid Tumors,Breast Cancer Glaxo Wellcome Prodrug of guanine Prodrug of arabinside TCell Leukemia/Lymphoma arabinside & B Cell Neoplasm Bristol Myers TaxaneAnalog Taxane Analog Taxol follow up Squib

In one embodiment, the methods of the invention use immunostimulatorynucleic acids as a replacement to the use of IFNα therapy in thetreatment of cancer. Currently, some treatment protocols call for theuse of IFNα. Since IFNα is produced following the administration of someimmunostimulatory nucleic acids, these nucleic acids can be used togenerate IFNα endogenously.

In another embodiment, the asthma/allergy medicament is a medicamentselected from the group consisting of PDE-4 inhibitor,bronchodilator/beta-2 agonist, K+ channel opener, VLA-4 antagonist,neurokin antagonist, TXA2 synthesis inhibitor, xanthanine, arachidonicacid antagonist, 5 lipoxygenase inhibitor, thromboxin A2 receptorantagonist, thromboxane A2 antagonist, inhibitor of 5-lipox activationprotein, and protease inhibitor, but is not so limited. In someimportant embodiments, the asthma/allergy medicament is abronchodilator/beta-2 agonist selected from the group consisting ofsalmeterol, salbutamol, terbutaline, D2522/formoterol, fenoterol, andorciprenaline.

In another embodiment, the asthma/allergy medicament is a medicamentselected from the group consisting of anti-histamines and prostaglandininducers. In one embodiment, the anti-histamine is selected from thegroup consisting of loratidine, cetirizine, buclizine, ceterizineanalogues, fexofenadine, terfenadine, desloratadine, norastemizole,epinastine, ebastine, ebastine, astemizole, levocabastine, azelastine,tranilast, terfenadine, mizolastine, betatastine, CS 560, and HSR 609.In another embodiment, the prostaglandin inducer is S-5751.

In yet another embodiment, the asthma/allergy medicament is selectedfrom the group consisting of steroids and immunomodulators. Theimmunomodulators may be selected from the group consisting ofanti-inflammatory agents, leukotriene antagonists, IL-4 muteins, solubleIL-4 receptors, immunosuppressants, anti-IL-4 antibodies, IL-4antagonists, anti-IL-5 antibodies, soluble IL-13 receptor-Fc fusionproteins, anti-IL-9 antibodies, CCR3 antagonists, CRY5 antagonists,VLA-4 inhibitors, and downregulators of IgE, but are not so limited. Inone embodiment, the downregulator of IgE is an anti-IgE. In anotherembodiment, the steroid is selected from the group consisting ofbeclomethasone, fluticasone, tramcinolone, budesonide, and budesonide.In still a further embodiment, the immunosuppressant is a tolerizingpeptide vaccine.

In one embodiment, the immunostimulatory nucleic acid is administeredconcurrently with the asthma/allergy medicament. In another embodiment,the subject is an immunocompromised subject.

Immunostimulatory nucleic acids can be combined with yet othertherapeutic agents such as adjuvants to enhance immune responses. Theimmunostimulatory nucleic acid and other therapeutic agent may beadministered simultaneously or sequentially. When the other therapeuticagents are administered simultaneously they can be administered in thesame or separate formulations, but are administered at the same time.The other therapeutic agents are administered sequentially with oneanother and with immunostimulatory nucleic acid, when the administrationof the other therapeutic agents and the immunostimulatory nucleic acidis temporally separated. The separation in time between theadministration of these compounds may be a matter of minutes or it maybe longer. Other therapeutic agents include but are not limited toadjuvants, cytokines, antibodies, antigens, etc.

The compositions of the invention may also comprise a non-nucleic acidadjuvants. A non-nucleic acid adjuvant is any molecule or compoundexcept for the immunostimulatory nucleic acids described herein whichcan stimulate the humoral and/or cellular immune response. Non-nucleicacid adjuvants include, for instance, adjuvants that create a depoteffect, immune stimulating adjuvants, and adjuvants that create a depoteffect and stimulate the immune system.

An adjuvant that creates a depot effect as used herein is an adjuvantthat causes the antigen to be slowly released in the body, thusprolonging the exposure of immune cells to the antigen. This class ofadjuvants includes but is not limited to alum (e.g., aluminum hydroxide,aluminum phosphate); or emulsion-based formulations including mineraloil, non-mineral oil, water-in-oil or oil-in-water-in oil emulsion,oil-in-water emulsions such as Seppic ISA series of Montanide adjuvants(e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (asqualene-in-water emulsion stabilized with Span 85 and Tween 80; ChironCorporation, Emeryville, Calif.; and PROVAX (an oil-in-water emulsioncontaining a stabilizing detergent and a micelle-forming agent; IDEC,Pharmaceuticals Corporation, San Diego, Calif.).

An immune stimulating adjuvant is an adjuvant that causes activation ofa cell of the immune system. It may, for instance, cause an immune cellto produce and secrete cytokines. This class of adjuvants includes butis not limited to saponins purified from the bark of the Q. saponariatree, such as QS21 (a glycolipid that elutes in the 21^(st) peak withHPLC fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.);poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus ResearchInstitute, USA); derivatives of lipopolysaccharides such asmonophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton,Mont.), muramyl dipeptide (MDP; Ribi) and threonyl-muramyl dipeptide(t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to lipid A; OMPharma SA, Meyrin, Switzerland); and Leishmania elongation factor (apurified Leishmania protein; Corixa Corporation, Seattle, Wash.).

Adjuvants that create a depot effect and stimulate the immune system arethose compounds which have both of the above-identified functions. Thisclass of adjuvants includes but is not limited to ISCOMS(immunostimulating complexes which contain mixed saponins, lipids andform virus-sized particles with pores that can hold antigen; CSL,Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2which is an oil-in-water emulsion containing MPL and QS21: SmithKlineBeecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKlineBeecham adjuvant system #4 which contains alum and MPL; SBB, Belgium);non-ionic block copolymers that form micelles such as CRL 1005 (thesecontain a linear chain of hydrophobic polyoxypropylene flanked by chainsof polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex AdjuvantFormulation (SAF, an oil-in-water emulsion containing Tween 80 and anonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).

The immunostimulatory nucleic acids are themselves useful as adjuvantsfor inducing a humoral immune response. Thus they can be delivered to asubject exposed to an antigen to produce an enhanced immune response tothe antigen.

The immunostimulatory nucleic acids are useful as mucosal adjuvants. Ithas previously been discovered that both systemic and mucosal immunityare induced by mucosal delivery of CpG nucleic acids. The systemicimmunity induced in response to CpG nucleic acids included both humoraland cell-mediated responses to specific antigens that were not capableof inducing systemic immunity when administered alone to the mucosa.Furthermore, both CpG nucleic acids and cholera toxin (CT, a mucosaladjuvant that induces a Th2-like response) induced CTL. This wassurprising since with systemic immunization, the presence of Th2-likeantibodies is normally associated with a lack of CTL (Schirmbeck et al.,1995). Based on the results presented herein it is expected that theimmunostimulatory nucleic acids will function in a similar manner.

Additionally, the immunostimulatory nucleic acids induce a mucosalresponse at both local (e.g., lung) and remote (e.g., lower digestivetract) mucosal sites. Significant levels of IgA antibodies are inducedat distant mucosal sites by the immunostimulatory nucleic acids. CT isgenerally considered to be a highly effective mucosal adjuvant. As hasbeen previously reported (Snider 1995), CT induces predominantly IgG1isotype of antibodies, which are indicative of Th2-type response. Incontrast, the immunostimulatory nucleic acids are more Th1 withpredominantly IgG2a antibodies, especially after boost or when the twoadjuvants are combined. Th1-type antibodies in general have betterneutralizing capabilities, and furthermore, a Th2 response in the lungis highly undesirable because it is associated with asthma (Kay, 1996,Hogg, 1997). Thus the use of immunostimulatory nucleic acids as amucosal adjuvant has benefits that other mucosal adjuvants cannotachieve. The immunostimulatory nucleic acids of the invention also areuseful as mucosal adjuvants for induction of both a systemic and amucosal immune response.

Mucosal adjuvants referred to as non-nucleic acid mucosal adjuvants mayalso be administered with the immunostimulatory nucleic acids. Anon-nucleic acid mucosal adjuvant as used herein is an adjuvant otherthan a immunostimulatory nucleic acid that is capable of inducing amucosal immune response in a subject when administered to a mucosalsurface in conjunction with an antigen. Mucosal adjuvants include butare not limited to Bacterial toxins e.g., Cholera toxin (CT), CTderivatives including but not limited to CT B subunit (CTB) (Wu et al.,1998, Tochikubo et al., 1998); CTD53 (Val to Asp) (Fontana et al.,1995); CTK97 (Val to Lys) (Fontana et al., 1995); CTK104 (Tyr to Lys)(Fontana et al., 1995); CTD53/K63 (Val to Asp, Ser to Lys) (Fontana etal., 1995); CTH54 (Arg to His) (Fontana et al., 1995); CTN₁₀₇ (His toAsn) (Fontana et al., 1995); CTE114 (Ser to Glu) (Fontana et al., 1995);CTE112K (Glu to Lys) (Yamamoto et al., 1997a); CTS61F (Ser to Phe)(Yamamoto et al., 1997a, 1997b); CTS106 (Pro to Lys) (Douce et al.,1997, Fontana et al., 1995); and CTK63 (Ser to Lys) (Douce et al., 1997,Fontana et al., 1995), Zonula occludens toxin, zot, Escherichia coliheat-labile enterotoxin, Labile Toxin (LT), LT derivatives including butnot limited to LT B subunit (LTB) (Verweij et al., 1998); LT7K (Arg toLys) (Komase et al., 1998, Douce et al., 1995); LT61F (Ser to Phe)(Komase et al., 1998); LT112K (Glu to Lys) (Komase et al., 1998); LT118E(Gly to Glu) (Komase et al., 1998); LT146E (Arg to Glu) (Komase et al.,1998); LT192G (Arg to Gly) (Komase et al., 1998); LTK63 (Ser to Lys)(Marchetti et al., 1998, Douce et al., 1997, 1998, Di Tommaso et al.,1996); and LTR72 (Ala to Arg) (Giuliani et al., 1998), Pertussis toxin,PT. (Lycke et al., 1992, Spangler B D, 1992, Freytag and Clemments,1999, Roberts et al., 1995, Wilson et al., 1995) including PT-9K/129G(Roberts et al., 1995, Cropley et al., 1995); Toxin derivatives (seebelow) (Holmgren et al., 1993, Verweij et al., 1998, Rappuoli et al.,1995, Freytag and Clements, 1999); Lipid A derivatives (e.g.,monophosphoryl lipid A, MPL) (Sasaki et al., 1998, Vancott et al., 1998;Muramyl Dipeptide (MDP) derivatives (Fukushima et al., 1996, Ogawa etal., 1989, Michalek et al., 1983, Morisaki et al., 1983); Bacterialouter membrane proteins (e.g., outer surface protein A (OspA)lipoprotein of Borrelia burgdorferi, outer membrane protine of Neisseriameningitidis) (Marinaro et al., 1999, Van de Verg et al., 1996);Oil-in-water emulsions (e.g., MF59) (Barchfield et al., 1999, Verschooret al., 1999, O'Hagan, 1998); Aluminum salts (Isaka et al., 1998, 1999);and Saponins (e.g., QS21) Antigenics, Inc., Woburn, Mass.) (Sasaki etal., 1998, MacNeal et al., 1998), ISCOMS, MF-59 (a squalene-in-wateremulsion stabilized with Span 85 and Tween 80; Chiron Corporation,Emeryville, Calif.); the Seppic ISA series of Montanide adjuvants (e.g.,Montanide ISA 720; AirLiquide, Paris, France); PROVAX (an oil-in-wateremulsion containing a stabilizing detergent and a micelle-forming agent;IDEC Pharmaceuticals Corporation, San Diego, Calif.); Syntex AdjuvantFormulation (SAF; Syntex Chemicals, Inc., Boulder, Colo.);poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus ResearchInstitute, USA) and Leishmania elongation factor (Corixa Corporation,Seattle, Wash.).

Immune responses can also be induced or augmented by theco-administration or co-linear expression of cytokines (Bueler &Mulligan, 1996; Chow et al., 1997; Geissler et al., 1997; Iwasaki etal., 1997; Kim et al., 1997) or B-7 co-stimulatory molecules (Iwasaki etal., 1997; Tsuji et al., 1997) with the immunostimulatory nucleic acids.The cytokines can be administered directly with immunostimulatorynucleic acids or may be administered in the form of a nucleic acidvector that encodes the cytokine, such that the cytokine can beexpressed in vivo. In one embodiment, the cytokine is administered inthe form of a plasmid expression vector. The term cytokine is used as ageneric name for a diverse group of soluble proteins and peptides whichact as humoral regulators at nano- to picomolar concentrations andwhich, either under normal or pathological conditions, modulate thefunctional activities of individual cells and tissues. These proteinsalso mediate interactions between cells directly and regulate processestaking place in the extracellular environment.

Examples of cytokines include, but are not limited to IL-1, IL-2, IL-4,IL-5, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, granulocyte-macrophagecolony stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), interferon-γ (γ-IFN), IFN-α, tumor necrosis factor(TNF), TGF-β, FLT-3 ligand, and CD40 ligand.

Cytokines play a role in directing the T cell response. Helper (CD4+) Tcells orchestrate the immune response of mammals through production ofsoluble factors that act on other immune system cells, including other Tcells. Most mature CD4+ T helper cells express one of two cytokineprofiles: Th1 or Th2. The Th1 subset promotes delayed-typehypersensitivity, cell-mediated immunity, and immunoglobulin classswitching to IgG_(2a). The Th2 subset induces humoral immunity byactivating B cells, promoting antibody production, and inducing classswitching to IgG₁ and IgE. In some embodiments, it is preferred that thecytokine be a Th1 cytokine.

The immunostimulatory nucleic acids may be directly administered to thesubject or may be administered in conjunction with a nucleic aciddelivery complex. A nucleic acid delivery complex shall mean a nucleicacid molecule associated with (e.g. ionically or covalently bound to; orencapsulated within) a targeting means (e.g. a molecule that results inhigher affinity binding to target cell (e.g., B cell surfaces and/orincreased cellular uptake by target cells). Examples of nucleic aciddelivery complexes include nucleic acids associated with a sterol (e.g.cholesterol), a lipid (e.g. a cationic lipid, virosome or liposome), ora target cell specific binding agent (e.g. a ligand recognized by targetcell specific receptor). Preferred complexes may be sufficiently stablein vivo to prevent significant uncoupling prior to internalization bythe target cell. However, the complex can be cleavable under appropriateconditions within the cell so that the nucleic acid is released in afunctional form.

Delivery vehicles or delivery devices for delivering antigen and nucleicacids to surfaces have been described. The Immunostimulatory nucleicacid and/or the antigen and/or other therapeutics may be administeredalone (e.g., in saline or buffer) or using any delivery vehicles knownin the art. For instance the following delivery vehicles have beendescribed: Cochleates (Gould-Fogerite et al., 1994, 1996); Emulsomes(Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993,Carlsson et al., 1991, Hu et., 1998, Morein et al., 1999); Liposomes(Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a,1995b); Live bacterial vectors (e.g., Salmonella, Escherichia coli,Bacillus calmatte-guerin, Shigella, Lactobacillus) (Hone et al., 1996,Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991,Nugent et al., 1998); Live viral vectors (e.g., Vaccinia, adenovirus,Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugentet al., 1998, Flexner et al., 1988, Morrow et al., 1999); Microspheres(Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore etal., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); Nucleic acidvaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998,Okada et al., 1997, Ishii et al., 1997); Polymers (e.g.carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill etal., 1998); Polymer rings (Wyatt et al., 1998); Proteosomes (Vancott etal., 1998, Lowell et al., 1988, 1996, 1997); Sodium Fluoride (Hashi etal., 1998); Transgenic plants (Tacket et al., 1998, Mason et al., 1998,Haq et al., 1995); Virosomes (Gluck et al., 1992, Mengiardi et al.,1995, Cryz et al., 1998); Virus-like particles (Jiang et al., 1999,Leibl et al., 1998). Other delivery vehicles are known in the art andsome additional examples are provided below in the discussion ofvectors.

The stimulation index of a particular immunostimulatory nucleic acid canbe tested in various immune cell assays. Preferably, the stimulationindex of the immunostimulatory nucleic acid with regard to B cellproliferation is at least about 5, preferably at least about 10, morepreferably at least about 15 and most preferably at least about 20 asdetermined by incorporation of ³H uridine in a murine B cell culture,which has been contacted with 20 μM of nucleic acid for 20 h at 37° C.and has been pulsed with 1 μCi of ³H uridine; and harvested and counted4 h later as described in detail in PCT Published Patent ApplicationsPCT/US95/01570 (WO 96/02555) and PCT/US97/19791 (WO 98/18810) claimingpriority to U.S. Ser. Nos. 08/386,063 and 08/960,774, filed on Feb. 7,1995 and Oct. 30, 1997 respectively. For use in vivo, for example, it isimportant that the immunostimulatory nucleic acids be capable ofeffectively inducing an immune response, such as, for example, antibodyproduction. Other assays designed to test efficacy and effective amountsare described in the Examples.

Immunostimulatory nucleic acids are effective in non-rodent vertebrate.Different immunostimulatory nucleic acid can cause optimal immunestimulation depending on the type of subject and the sequence of theimmunostimulatory nucleic acid. Many vertebrates have been foundaccording to the invention to be responsive to the same class ofimmunostimulatory nucleic acids, sometimes referred to as human specificimmunostimulatory nucleic acids. Rodents, however, respond to differentnucleic acids. As shown herein an immunostimulatory nucleic acid causingoptimal stimulation in humans may not generally cause optimalstimulation in a mouse and vice versa. An immunostimulatory nucleic acidcausing optimal stimulation in humans often does, however, cause optimalstimulation in other animals such as cow, horses, sheep, etc. One ofskill in the art can identify the optimal nucleic acid sequences usefulfor a particular species of interest using routine assays describedherein and/or known in the art, using the guidance supplied herein.

The term effective amount of a immunostimulatory nucleic acid refers tothe amount necessary or sufficient to realize a desired biologic effect.For example, an effective amount of a immunostimulatory nucleic acid forinducing mucosal immunity is that amount necessary to cause thedevelopment of IgA in response to an antigen upon exposure to theantigen, whereas that amount required for inducing systemic immunity isthat amount necessary to cause the development of IgG in response to anantigen upon exposure to the antigen. Combined with the teachingsprovided herein, by choosing among the various active compounds andweighing factors such as potency, relative bioavailability, patient bodyweight, severity of adverse side-effects and preferred mode ofadministration, an effective prophylactic or therapeutic treatmentregimen can be planned which does not cause substantial toxicity and yetis entirely effective to treat the particular subject. The effectiveamount for any particular application can vary depending on such factorsas the disease or condition being treated, the particularimmunostimulatory nucleic acid being administered, the antigen, the sizeof the subject, or the severity of the disease or condition. One ofordinary skill in the art can empirically determine the effective amountof a particular immunostimulatory nucleic acid and/or antigen and/orother therapeutic agent without necessitating undue experimentation.

Subject doses of the compounds described herein for mucosal or localdelivery typically range from about 0.1 μg to 10 mg per administration,which depending on the application could be given daily, weekly, ormonthly and any other amount of time therebetween. More typicallymucosal or local doses range from about 10 μg to 5 mg peradministration, and most typically from about 100 μg to 1 mg, with 2-4administrations being spaced days or weeks apart. More typically, immunestimulant doses range from 1 μg to 10 mg per administration, and mosttypically 10 μg to 1 mg, with daily or weekly administrations. Subjectdoses of the compounds described herein for parenteral delivery for thepurpose of inducing an antigen-specific immune response, wherein thecompounds are delivered with an antigen but not another therapeuticagent are typically 5 to 10,000 times higher than the effective mucosaldose for vaccine adjuvant or immune stimulant applications, and moretypically 10 to 1,000 times higher, and most typically 20 to 100 timeshigher. Doses of the compounds described herein for parenteral deliveryfor the purpose of inducing an innate immune response or for increasingADCC or for inducing an antigen specific immune response when theimmunostimulatory nucleic acids are administered in combination withother therapeutic agents or in specialized delivery vehicles typicallyrange from about 0.1 μg to 10 mg per administration, which depending onthe application could be given daily, weekly, or monthly and any otheramount of time therebetween. More typically parenteral doses for thesepurposes range from about 10 μg to 5 mg per administration, and mosttypically from about 100 μg to 1 mg, with 2-4 administrations beingspaced days or weeks apart. In some embodiments, however, parenteraldoses for these purposes may be used in a range of 5 to 10,000 timeshigher than the typical doses described above.

For any compound described herein the therapeutically effective amountcan be initially determined from animal models. A therapeuticallyeffective dose can also be determined from human data for CpGoligonucleotides which have been tested in humans (human clinical trialshave been initiated) and for compounds which are known to exhibitsimilar pharmacological activities, such as other mucosal adjuvants,e.g., LT and other antigens for vaccination purposes, for the mucosal orlocal administration. Higher doses are required for parenteraladministration. The applied dose can be adjusted based on the relativebioavailability and potency of the administered compound. Adjusting thedose to achieve maximal efficacy based on the methods described aboveand other methods as are well-known in the art is well within thecapabilities of the ordinarily skilled artisan.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

For use in therapy, an effective amount of the immunostimulatory nucleicacid can be administered to a subject by any mode that delivers thenucleic acid to the desired surface, e.g., mucosal, systemic.Administering the pharmaceutical composition of the present inventionmay be accomplished by any means known to the skilled artisan. Preferredroutes of administration include but are not limited to oral,parenteral, intramuscular, intranasal, intratracheal, inhalation,ocular, vaginal, and rectal.

For oral administration, the compounds (i.e., immunostimulatory nucleicacids, antigens and other therapeutic agents) can be formulated readilyby combining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a subject to be treated. Pharmaceutical preparations fororal use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer, Science 249:1527-1533,1990, which is incorporated herein by reference.

The immunostimulatory nucleic acids and optionally other therapeuticsand/or antigens may be administered per se (neat) or in the form of apharmaceutically acceptable salt. When used in medicine the salts shouldbe pharmaceutically acceptable, but non-pharmaceutically acceptablesalts may conveniently be used to prepare pharmaceutically acceptablesalts thereof. Such salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

As described in greater detail herein, the pharmaceutical compositionsof the invention contain an effective amount of a immunostimulatorynucleic acid and optionally antigens and/or other therapeutic agentsoptionally included in a pharmaceutically-acceptable carrier. The termpharmaceutically-acceptable carrier means one or more compatible solidor liquid filler, diluents or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm carrier denotes an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application. The components of the pharmaceutical compositions alsoare capable of being commingled with the compounds of the presentinvention, and with each other, in a manner such that there is nointeraction which would substantially impair the desired pharmaceuticalefficiency.

The immunostimulatory nucleic acids useful in the invention may bedelivered in mixtures with additional adjuvant(s), other therapeutics,or antigen(s). A mixture may consist of several adjuvants in addition tothe immunostimulatory nucleic acid or several antigens or othertherapeutics.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular adjuvants orantigen selected, the particular condition being treated and the dosagerequired for therapeutic efficacy. The methods of this invention,generally speaking, may be practiced using any mode of administrationthat is medically acceptable, meaning any mode that produces effectivelevels of an immune response without causing clinically unacceptableadverse effects. Preferred modes of administration are discussed above.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the compounds into associationwith a carrier which constitutes one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the compounds into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct. Liquid dose units are vials or ampoules. Solid dose units aretablets, capsules and suppositories. For treatment of a patient,depending on activity of the compound, manner of administration, purposeof the immunization (i.e., prophylactic or therapeutic), nature andseverity of the disorder, age and body weight of the patient, differentdoses may be necessary. The administration of a given dose can becarried out both by single administration in the form of an individualdose unit or else several smaller dose units. Multiple administration ofdoses at specific intervals of weeks or months apart is usual forboosting the antigen-specific responses.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. They include polymerbase systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides.

Microcapsules of the foregoing polymers containing drugs are describedin, for example, U.S. Pat. No. 5,075,109. Delivery systems also includenon-polymer systems that are: lipids including sterols such ascholesterol, cholesterol esters and fatty acids or neutral fats such asmono-di-and tri-glycerides; hydrogel release systems; sylastic systems;peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775, 4,675,189,and 5,736,152, and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-basedhardware delivery systems can be used, some of which are adapted forimplantation.

The present invention is further illustrated by the following Examples,which in no way should be construed as further limiting. The entirecontents of all of the references (including literature references,issued patents, published patent applications, and co-pending patentapplications) cited throughout this application are hereby expresslyincorporated by reference.

EXAMPLES

Summary:

The in vitro ability of ODN 10103 (SEQ ID NO:1) to stimulate human PBMCwas compared to that of ODN 7909 (SEQ ID NO:2). Immune stimulation wasanalyzed in terms of receptor (i.e., TLR9) engagement, B cell activation(e.g., expression of cell surface activation markers and B cellproliferation), and cytokine secretion (e.g., secretion of IL-10, IP-10,IFN-α and TNF-α). All assays demonstrated that ODN 10103 has propertiessimilar to or superior to those of ODN 7909.

The ability of ODN 10103 to stimulate murine immune cells in vitro andin vivo was compared to that of ODN 7909. In vitro studies (e.g., B cellproliferation assays, NK lytic activity, and cytokine secretionprofiles) were carried out using naïve BALB/c mouse splenocytes. In vivostudies were carried out by examining the potential of these two ODNs toenhance antigen specific immune responses to hepatitis B surface antigen(HBsAg), with both humoral (antibody) and cell mediated immune responses(CTL activity) analyzed. In addition, the Th-bias of the induced immuneresponse was examined by determining the strength of the CTL response aswell as the IgG2a/IgG1 ratio.

Materials and Methods:

Oligodeoxynucleotides: All ODNs (10103, 7909 and a control) wereprovided by Coley Pharmaceutical GmbH (Langenfeld, Germany). The controlODN contained no stimulatory CpG motif. ODNs were diluted inphosphate-buffered saline, and stored at −20° C. All dilutions werecarried out using pyrogen-free reagents.TLR9 assay: Cells used for this assay expressed the human TLR9 receptorand contained a reporter gene construct. Cells were incubated with ODNsfor 16 h. Each data point was done in triplicate. Cells were lysed andassayed for reporter gene activity. Stimulation indices were calculatedin reference to reporter gene activity of medium without addition ofODN.Cell purification: Peripheral blood buffy coat preparations from healthyhuman donors were obtained from the German Red Cross (Rathingen,Germany) and from these, PBMC were purified by centrifugation overFicoll-Hypaque (Sigma, Germany). The purified PBMC were either usedfresh or were suspended in freezing medium and stored at −70° C. Whenrequired, aliquots of these cells were thawed, washed and resuspended inRPMI 1640 culture medium supplemented with 10% (v/v) heat inactivatedFCS, 1.5 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.Cytokine detection: Thawed or fresh PBMC were resuspended at aconcentration of 5×10⁶/ml and added to 48 well flat-bottomed plates (1ml/well), which had previously received nothing or ODN in a variety ofconcentrations. The cells were cultured in a humidified incubator at 37°C. Culture supernatants were collected after the indicated time points.If not used immediately, supernatants were frozen at −20° C. untilrequired.

Amounts of cytokines in the supernatants were assessed usingcommercially available ELISA Kits (IL-10; Diaclone, USA) or in-houseELISAs (IP-10 and IFN-α) developed using commercially availableantibodies (from Pharmingen or PBL; Germany or USA, respectively).

Cultures for flow cytometric analysis of B cell activation: Monoclonalantibodies to CD19 and CD86 were purchased from Becton Dickinson(Germany). PBMC were incubated for 48 hours with or without the additionof different concentrations of ODNs. B cells were identified byexpression of CD19 by flow cytometry. Flow cytometric data were acquiredon a FACSCalibur (Becton Dickinson). Data were analyzed using thecomputer program CellQuest (Becton Dickinson). Proliferating CD19positive B cells were identified after culturing CFSE-labelled PBMC(CFSE is a fluorescing dye binding to all cell surfaces) by decreasedCFSE content using flow cytometry methodology (see above).Murine In Vitro/In Vivo Studies:Oligodeoxynucleotides: CpG ODN 7909 and CpG ODN 10103 were both used inGMP quality forms. Quality control of CpG ODN 7909 and CpG ODN 10103 wascarried out by Coley Pharmaceutical GmbH (Langenfeld, Germany). All ODNwere resuspended in sterile, endotoxin free TE at pH 8.0 (OmniPer®; EMScience, Gibbstown, N.J.) and stored and handle under aseptic conditionsto prevent both microbial and endotoxin contamination. Dilution of ODNsfor assays was carried out in sterile, endotoxin free PBS, pH 7.2 (SigmaChemical Company, St. Lois, Mo.).Animals: Female BALB/c mice (6-8 weeks of age) were used for allexperiments. Animals were purchased from Charles River Canada (Quebec,Canada) and housed in micro isolators at the animal care facility of theOttawa Hospital Research Institute, Civic Site.Splenocyte harvest and culture: Naïve BALB/c mouse splenocytes were usedfor all in vitro assays. Animals were anesthetized with isofluorane andeuthanized by cervical dislocation. Spleens were removed under asepticconditions and placed in PBS+0.2% bovine serum albumin (Sigma ChemicalCompany). Spleens were then homogenized and splenocytes werere-suspended in RPMI 1640 (Life Technologies, Grand Island, N.Y.) tissueculture medium supplemented with 2% normal mouse serum (CedarlaneLaboratories, Ontario, Canada), penicillin-streptomycin solution (finalconcentration of 1000 U/ml and 1 mg/ml respectively; Sigma ChemicalCompany), and 5×10⁻⁵ M β-mercaptoethanol (Sigma Chemical Company).B cell proliferation assays: Spleen cell suspensions were prepared andadjusted to a final concentration of 5×10⁶ cells per ml in complete RPMI1640. Splenocyte suspension was plated onto 96-well U-bottom tissueculture plates (100 μl/well) along with 100 μl of each stimulant dilutedto appropriate concentrations in complete RPMI 1640. The stimulants usedwere CpG ODN (at 1, 3, 10 μg/ml) 7909 and 10103. Concanavalin A (10μg/ml, Sigma Chemical Company) LPS (10 μg/ml, Sigma Chemical Company)were used as a positive controls and cells cultured with media alonewere used as negative controls. Each splenocyte sample was plated intriplicate and the cells were incubated in a humidified 5% CO₂ incubatorat 37° C. for 96 hr. At the end of the incubation period, cells werepulsed with ³H-thymidine (20 μCi/ml) at 96 hr post incubation for 16hours, harvested and measured for radioactivity.Cytokine secretion profiles: Spleen cell suspensions were prepared andplated in 96-well U-bottom tissue culture plates as described for B cellproliferation assays. Each splenocyte sample was plated in triplicateand the cells were incubated in a humidified 5% CO₂ incubator at 37° C.for either 6, 12 or 48 hr. At the end of the incubation period, the96-well plates were centrifuged for 5 min at 1200 rpm and culturesupernatants harvested and stored at −80° C. until assayed. Commerciallyavailable assay kits (mouse OptEIA kits; PharMingen, Mississauga, ON)were used according to manufacturers instructions to assay cytokinelevels in culture supernatants taken at 6 hr (TNF-α), 24 hr (IL-12) and48 hr (IL-6 and IL-10).NK assays: Splenocyte suspensions were prepared as described previouslyand adjusted to a final concentration of 3×10⁶ cells per ml in completeRPMI 1640. Splenocyte suspension (10 ml; 30×10⁶ cells) was plated inT-25 tissue culture flasks (Fisher Scientific, Ottawa, ON) along witheither CpG ODN (at 1, 3, 10 μg/ml) 7909 and 10103. Splenocytes culturedwith media alone was used as negative controls. Each splenocyte culturewas incubated in a humidified 5% CO₂ incubator at 37° C. for 24 hr. Atthe end of the incubation period, cells were plated at differenteffector:target ratios onto 96-well U-bottom tissue culture plates (100μl/well) along with 100 μl of ⁵¹Cr labeled target cells at 5×10⁴cells/ml. NK sensitive mouse lymphoma cell line YAC-1 (ATCC # TIB-160,ATCC, Manassas, Va.) was used as the target cell line. Each sample wasplated in triplicate and the cells were incubated in a humidified 5% CO₂incubator at 37° C. for 4 hr. Target cells were incubated with mediaalone or with 2N HCl to determine spontaneous release and maximumrelease respectively. At the end of the incubation period, supernatantswere harvested and radioactivity levels were determined using a γcounter. The % lysis was determined using the following formula:

${\%\mspace{14mu}{specific}\mspace{14mu}{release}} = {\frac{\begin{matrix}{{{experimental}\mspace{14mu}{release}} -} \\{{spontaneous}\mspace{14mu}{release}}\end{matrix}}{\begin{matrix}{{{maximum}\mspace{14mu}{release}} -} \\{{spontaneous}\mspace{14mu}{release}}\end{matrix}} \times 100}$Immunization of mice: BALB/c mice (n=10/group) were immunized with 1 μgHBsAg sub type ad (International Enzymes, Calif.) alone or incombination with either 10 μg CpG ODN 7909 or CpG ODN 10103. Animalswere bled and boosted at 4 weeks post-primary immunization. At 1 weekpost boost 5 animals from each group was euthanized and spleens removedfor CTL assays.Determination of antibody responses: Antibodies (total IgG, IgG1 andIgG2a) specific to HBsAg (anti-HBs) were detected and quantified byendpoint dilution ELISA assay, which was performed in triplicate onsamples from individual animals (2). End-point titers were defined asthe highest plasma dilution that resulted in an absorbance value (OD450) two times greater than that of non-immune plasma with a cut-offvalue of 0.05. These were reported as group mean titers ±SEM.Evaluation of CTL responses: CTL assays were conducted as previouslydescribed (3). The results are presented as % specific lysis atdifferent effector: target (E:T) ratios.Statistical analysis: Statistical analysis was performed using InStatprogram (Graph PAD Software, San Diego). The statistical differencebetween groups were determined by Student's t test (for two groups) orby 1-factor ANOVA followed by Tukey's test (for three or more groups) onraw data or transformed data (log₁₀, for heteroscedastic populations).Results:TLR9 engagement: Recently the receptor for the recognition of CpGsequences was identified and shown to be a member of the Toll-LikeReceptor (TLR) family (Hemmi et al., 2000). This receptor, TLR9, isreadily activated by ODNs containing optimal immunostimulatory CpGsequences. We incubated a cell line stably expressing the human TLR9with different concentrations of ODNs 7909 and 10103 as well as acontrol ODN (FIG. 1).

Both ODNs showed a concentration dependent dose-response curve reachingtheir maximum activation at the same concentration. A control ODN didnot induce TLR9 activation even at the highest concentration of 24μg/ml. ODN 10103 showed higher stimulation capacity at lower doses thandid ODN 7909 (e.g., at 6 and 12 μg/ml), suggesting that ODN 10103 can beused at lower doses to achieve similar immunostimulation indices, andthereby reducing potential toxicity.

Human B cells: One characteristic of type B ODNs is their ability tovery efficiently activate B cells (Krieg et al., 1995). B cells andplasmacytoid DC are at the moment the only immune cell types known toexpress TLR9 (Krug et al., 2001; Bauer et al., 2001). We, therefore,measured the direct activation of B cells induced by ODNs 7909 and 10103by up regulation of the cell surface marker CD86 (FIG. 2), and measuringthe proliferation of B cells (FIG. 3). For CD86 expression on human Bcells PBMC of healthy blood donors were incubated with different ODNsand B cell activation measured as described in Materials and Methods.

Both results demonstrate that 10103 at least as well as 7909 as astimulator of human B cells. FIG. 2 shows that these CpG ODNs were ableto stimulate B cells starting at an in vitro concentration of only 0.4μg/ml. The plateau was reached at about 1.6 μg/ml and more than 60% of Bcells were found to have up regulated CD86 in contrast to the controlthat was much less potent at the same concentration. The resultsindicate that ODN 10103 stimulates CD86 expression to a higher level ata lower dose than dose ODN 7909 (e.g., at 0.4 μg/ml) in all three donorstested, again suggesting that smaller doses of ODN 10103 could be usedto achieve desired levels of immunostimulation. A similar result wasobtained for the induction of B cell proliferation (FIG. 3).

Cytokine secretion: ODNs of the B class lead to a Th1 dominated immuneresponse in vivo as well as in vitro. It was found that they are able toinduce typical Th1 cytokines such as IFN-γ and IFN-α as well aschemokines such as MCP-1 and IP-10. In addition, low secretion of thepro-inflammatory cytokines IL-6 as well as TNF-α and secretion of thenegative regulator IL-10 can be observed. We, therefore, measured thesecretion of the Th1 cytokine IFN-α, the chemokine IP-10 as well as theregulatory cytokine IL-10 and pro-inflammatory cytokine TNF-α.

FIG. 4 shows the result for an experiment performed with 6 differentdonors at 0.2, 0.4 and 1.6 μg/ml to measure in vitro IFN-α secretion.

Both CpG ODNs, 7909 as well as 10103, induced significant amounts ofIFN-α in different donors. In contrast, the control ODN induced no orlow amounts of IFN-α in one donor. The data suggests that a patientvariability may exist with some patients responding better to ODNs suchas 10103, as compared to ODN 7909. This finding indicates that ODN maybe classified in terms of the subjects that are likely to be highresponders.

In addition to IFN-α, ODNs 7909 and 10103 induced chemokine IP-10 asshown in FIG. 5. ODN 10103 induced equal or higher levels of IP-10 thandid ODN 7909, at all doses tested. In particular, at the 0.4 μg/mlconcentration, ODN 7909 produced higher amounts of IP-10 than did ODN7909. At a 1.6 μg/ml concentration, ODN 10103 induced roughly 25% moreIP-10 than did ODN 7909.

As demonstrated in FIG. 6, CpG ODNs 7909 and 10103 demonstrated almostidentical IL-10 induction capacity.

As shown in FIG. 7, both ODNs 7909 and 10103 as well as the control ODNshowed a low secretion profile of the pro-inflammatory cytokine TNF-α inall tested concentrations in comparison to LPS. At the highest dosetested (i.e., 6 μg/ml), ODN 7909 stimulated the secretion of higherlevels of IL-10 than did ODN 7909. The dose responses are shown in FIG.9.

In vitro mouse studies: According to the data, both CpG ODN 7909 and10103 are equally potent in inducing mouse B cell proliferation, haveessentially equal potency in enhancing cytokine secretion by mousesplenocytes, and have essentially equal potency in enhancing lyticactivity of NK cells in mouse splenocyte cultures (FIG. 10). ODN 10103appears to have a higher capacity for stimulating secretion of IL-6 andTNF-, particularly at the lower doses tested. Similary, the lyticactivity profiles of these ODNs differ according to the concentration.In vivo mouse studies: According to the results of this study use ofeither CpG ODN 7909 or 10103 significantly enhanced antibody titersagainst HBsAg compared to antigen alone (p<0.001 and p<0.01respectively) whereas there was no significant increase in anti-HBsresponses when control ODN was used in combination with HBsAg (p=0.85).(See FIGS. 11 and 12)

Furthermore, both CpG ODN 7909 and 10103 were equally potent inenhancing antibody responses against HBsAg in that there was nosignificant difference in anti-HBs responses in mice immunized withHBsAg+CpG ODN 7909 and HBsAg+CpG ODN 10103 (p=0.13).

In mice IgG isotype distribution is widely used as an indication of thenature of the immune response where a high IgG2a/IgG1 ratios areindicative of a Th1 biased immune response (1). In the present study,the use of CpG ODN significantly enhanced IgG2a titers compared to whenantigen was used alone or in combination with control ODN 2137 (p<0.001for Ag vs. 7909 or 10103 and p<0.01 for Ag+7909 vs. Ag+2137 and p<0.05for Ag+10103 vs. Ag+2137). However, the level of IgG2a response wassimilar when either CpG ODN 7909 or 10103 was used in combination withHBsAg (p>0.05). Therefore, both CpG ODN 7909 and 10103 are equallypotent in their ability to induce Th1 biased immune responses asmeasured by the increased levels of IgG2a over IgG1.

The CTL responses in animals immunized with HBsAg using ODN 10103 appearto be greater than those induced CpG ODN 7909, as shown in FIG. 13.

CONCLUSIONS

The in vitro data on human PBMC demonstrates that the molecules of the Bclass (7909 and 10103) behave similarly but not identically in all ofthe assays performed. Of particular importance is the observation thatfor several of the tested assays and functionalities, the ODN 10103induced greater immunostimulation than did previously identified ODN7909. This difference suggests that CpG nucleotides can be tailored foruse in subjects, and also that lower levels of CpG ODNs can achievedesired therapeutic endpoints, with potentially lower toxicity events.

REFERENCES

-   1. Bauer, S. et al.; Human TLR9 confers responsiveness to bacterial    DNA via species-specific CpG motif recognition; PNAS 98, 2001.-   2. Constant, S. L., and K. Bottomly 1997. Induction of Th1 and Th2    CD4+ T cell responses: the alternative approaches Annu Rev Immunol.    15:297-322.-   3. Davis, H. L., R. Weeratna, T. J. Waldschmidt, L. Tygrett, J.    Schorr, and A. M. Krieg 1998. CpG DNA is a potent enhancer of    specific immunity in mice immunized with recombinant hepatitis B    surface antigen J Immunol. 160:870-6.-   4. Hemmi, H. et al.; A Toll-like receptor recognizes bacterial DNA;    Nature 408, 2000.-   5. Krieg, A. M. et al.; CpG motifs in bacterial DNA trigger direct    B-cell activation; Nature 374, 1995.-   6. Krug, A. et al.; Toll-like receptor expression reveals CpG DNA as    a unique microbial stimulus for pDC which synergizes with CD40    ligand to induce high amounts of IL-12; Eur. J. Immunol. 31; 2001.-   7. McCluskie, M. J., and H. L. Davis 1998. CpG DNA is a potent    enhancer of systemic and mucosal immune responses against hepatitis    B surface antigen with intranasal administration to mice J Immunol.    161:4463-6

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

1. A composition comprising an immunostimulatory nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, wherein theimmunostimulatory nucleic acid has a nucleotide backbone comprising atleast one phosphorothioate modification, and wherein theimmunostimulatory nucleic acid comprises a CpG motif which isunmethylated.
 2. The composition of claim 1, wherein theimmunostimulatory nucleic acid molecule consists of the nucleotidesequence of SEQ ID NO:1.
 3. The composition of claim 1, furthercomprising an antigen.
 4. The composition of claim 3, wherein theantigen is selected from the group consisting of a microbial antigen, acancer antigen, and an allergen.
 5. The composition of claim 4, whereinthe microbial antigen is selected from the group consisting of abacterial antigen, a viral antigen, a fungal antigen and a parasiticantigen.
 6. The composition of claim 3, wherein the antigen is encodedby a nucleic acid vector.
 7. The composition of claim 3, wherein thenucleic acid vector is separate from the immunostimulatory nucleic acid.8. The composition of claim 3, wherein the antigen is a peptide antigen.9. The composition of claim 1, further comprising an adjuvant.
 10. Thecomposition of claim 9, wherein the adjuvant is a mucosal adjuvant. 11.The composition of claim 1, further comprising a cytokine.
 12. Thecomposition of claim 1, further comprising a therapeutic agent selectedfrom the group consisting of an anti-microbial agent, an anti-canceragent and an allergy/asthma medicament.
 13. The composition of claim 12,wherein the anti-microbial agent is selected from the group consistingof an anti-bacterial agent, an anti-viral agent, an anti-fungal agent,and an anti-parasite agent.
 14. The composition of claim 12, wherein theanti-cancer agent is selected from the group consisting of achemotherapeutic agent, a cancer vaccine, and an immunotherapeuticagent.
 15. The composition of claim 12, wherein the allergy/asthmamedicament is selected from the group consisting of PDE-4 inhibitor,bronchodilator/beta-2 agonist, K+ channel opener, VLA-4 antagonist,neurokin antagonist, TXA2 synthesis inhibitor, xanthanine, arachidonicacid antagonist, 5 lipoxygenase inhibitor, thromboxin A2 receptorantagonist, thromboxane A2 antagonist, inhibitor of 5-lipox activationprotein, and protease inhibitor.
 16. The composition of claim 1, whereinthe nucleotide backbone is chimeric.
 17. The composition of claim 1,wherein the nucleotide backbone is entirely modified.
 18. Thecomposition of claim 1, further comprising a pharmaceutically acceptablecarrier.
 19. The composition of claim 1, wherein the immunostimulatorynucleic acid is free of methylated CpG dinucleotides.
 20. Thecomposition of claim 1, wherein the immunostimulatory nucleic acidincludes more than four CpG motifs.
 21. The composition of claim 1,wherein the immunostimulatory nucleic acid is T-rich.
 22. Thecomposition of claim 1, wherein the immunostimulatory nucleic acidincludes a poly-T sequence.
 23. The composition of claim 1, wherein theimmunostimulatory nucleic acid includes a poly-G sequence.
 24. Thecomposition of claim 1, wherein the immunostimulatory nucleic acid isformulated for oral administration.
 25. The composition of claim 1,wherein the immunostimulatory nucleic acid is formulated as anutritional supplement.
 26. The composition of claim 25, wherein thenutritional supplement is formulated as a capsule, a pill, or asublingual tablet.
 27. The composition of claim 1, wherein theimmunostimulatory nucleic acid is formulated for local administration.28. The composition of claim 1, wherein the immunostimulatory nucleicacid is formulated for parenteral administration.
 29. The composition ofclaim 1, wherein the immunostimulatory nucleic acid is formulated in asustained release device.
 30. The composition of claim 1, wherein theimmunostimulatory nucleic acid is formulated for delivery to a mucosalsurface.
 31. The composition of claim 1, wherein the mucosal surface isselected from the group consisting of an oral, nasal, rectal, vaginal,and ocular surface.
 32. The composition of claim 1, wherein theimmunostimulatory nucleic acid stimulates a mucosal immune response. 33.The composition of claim 1, wherein the immunostimulatory nucleic acidstimulates a systemic immune response.
 34. The composition of claim 1,wherein the immunostimulatory nucleic acid is provided in an amounteffective to stimulate a mucosal immune response.
 35. The composition ofclaim 1, wherein the immunostimulatory nucleic acid is provided in anamount effective to stimulate a systemic immune response.
 36. Thecomposition of claim 1, wherein the immunostimulatory nucleic acid isprovided in an amount effective to stimulate an immune response.
 37. Thecomposition of claim 1, wherein the immunostimulatory nucleic acid isprovided in an amount effective to stimulate an immune response againstan infectious agent.
 38. The composition of claim 1, wherein theimmunostimulatory nucleic acid is provided in an amount effective tostimulate an immune response against an allergen.
 39. The composition ofclaim 1, wherein the immunostimulatory nucleic acid is provided in anamount effective to stimulate an immune response against a cancer. 40.The composition of claim 29, wherein the sustained release device is amicroparticle.
 41. The composition of claim 37, wherein the infectiousagent is a herpes simplex virus.
 42. A method for stimulating an immuneresponse in a subject in need thereof comprising administering to asubject a composition comprising an immunostimulatory nucleic acidmolecule comprising the nucleotide sequence of SEQ ID NO:1, in an amounteffective to stimulate an immune response, wherein in theimmunostimulatory nucleic acid comprises a CpG motif which isunmethylated.
 43. A method for inducing an innate immune response,comprising administering to the subject a composition comprising animmunostimulatory nucleic acid comprising the nucleotide sequence of SEQID NO:1, in an amount effective for activating an innate immuneresponse, wherein the immunostimulatory nucleic acid comprises a CpGmotif which is unmethylated.
 44. A composition comprising animmunostimulatory nucleic acid comprising the nucleotide sequence of SEQID NO:1, wherein the immunostimulatory nucleic acid is 21-100nucleotides in length, and wherein the immunostimulatory nucleic acidcomprises a CpG motif which is unmethylated.
 45. A compositioncomprising an immunostimulatory nucleic acid comprising the nucleotidesequence of SEQ ID NO:1 and an antigen, wherein the immunostimulatorynucleic acid comprises a CpG motif which is unmethylated.
 46. Acomposition comprising an immunostimulatory nucleic acid comprising thenucleotide sequence of SEQ ID NO:1, wherein the immunostimulatorynucleic acid is single stranded and wherein the immunostimulatorynucleic acid comprises a CpG motif which is unmethylated.
 47. Acomposition comprising an immunostimulatory nucleic acid consisting ofthe nucleotide sequence of SEQ ID NO:1, wherein the immunostimulatorynucleic acid comprises a CpG motif which is unmethylated.
 48. The methodof claim 42, wherein the immunostimulatory nucleic acid is 21-100nucleotides in length.
 49. The method of claim 42, wherein theimmunostimulatory nucleic acid consists of the nucleotide sequence ofSEQ ID NO:1.
 50. The method of claim 42, wherein the compositioncomprises an antigen.